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5 Unethical Medical Experiments Brought Out of the Shadows of History

Prisoners and other vulnerable populations often bore the brunt of unethical medical experimentation..

Most people are aware of some of the heinous medical experiments of the past that violated human rights. Participation in these studies was either forced or coerced under false pretenses. Some of the most notorious examples include the experiments by the Nazis, the Tuskegee syphilis study, the Stanford Prison Experiment, and the CIA’s LSD studies.

But there are many other lesser-known experiments on vulnerable populations that have flown under the radar. Study subjects often didn’t — or couldn’t — give consent. Sometimes they were lured into participating with a promise of improved health or a small amount of compensation. Other times, details about the experiment were disclosed but the extent of risks involved weren’t.

This perhaps isn’t surprising, as doctors who conducted these experiments were representative of prevailing attitudes at the time of their work. But unfortunately, even after informed consent was introduced in the 1950s , disregard for the rights of certain populations continued. Some of these researchers’ work did result in scientific advances — but they came at the expense of harmful and painful procedures on unknowing subjects.

Here are five medical experiments of the past that you probably haven’t heard about. They illustrate just how far the ethical and legal guidepost, which emphasizes respect for human dignity above all else, has moved.

The Prison Doctor Who Did Testicular Transplants

From 1913 to 1951, eugenicist Leo Stanley was the chief surgeon at San Quentin State Prison, California’s oldest correctional institution. After performing vasectomies on prisoners, whom he recruited through promises of improved health and vigor, Stanley turned his attention to the emerging field of endocrinology, which involves the study of certain glands and the hormones they regulate. He believed the effects of aging and decreased hormones contributed to criminality, weak morality, and poor physical attributes. Transplanting the testicles of younger men into those who were older would restore masculinity, he thought.  

Stanley began by using the testicles of executed prisoners — but he ran into a supply shortage. He solved this by using the testicles of animals, including goats and deer. At first, he physically implanted the testicles directly into the inmates. But that had complications, so he switched to a new plan: He ground up the animal testicles into a paste, which he injected into prisoners’ abdomens. By the end of his time at San Quentin, Stanley did an estimated 10,000 testicular procedures .

The Oncologist Who Injected Cancer Cells Into Patients and Prisoners

During the 1950s and 1960s, Sloan-Kettering Institute oncologist Chester Southam conducted research to learn how people’s immune systems would react when exposed to cancer cells. In order to find out, he injected live HeLa cancer cells into patients, generally without their permission. When patient consent was given, details around the true nature of the experiment were often kept secret. Southam first experimented on terminally ill cancer patients, to whom he had easy access. The result of the injection was the growth of cancerous nodules , which led to metastasis in one person.

Next, Southam experimented on healthy subjects , which he felt would yield more accurate results. He recruited prisoners, and, perhaps not surprisingly, their healthier immune systems responded better than those of cancer patients. Eventually, Southam returned to infecting the sick and arranged to have patients at the Jewish Chronic Disease Hospital in Brooklyn, NY, injected with HeLa cells. But this time, there was resistance. Three doctors who were asked to participate in the experiment refused, resigned, and went public.

The scandalous newspaper headlines shocked the public, and legal proceedings were initiated against Southern. Some in the scientific and medical community condemned his experiments, while others supported him. Initially, Southam’s medical license was suspended for one year, but it was then reduced to a probation. His career continued to be illustrious, and he was subsequently elected president of the American Association for Cancer Research.

The Aptly Named ‘Monster Study’

Pioneering speech pathologist Wendell Johnson suffered from severe stuttering that began early in his childhood. His own experience motivated his focus on finding the cause, and hopefully a cure, for stuttering. He theorized that stuttering in children could be impacted by external factors, such as negative reinforcement. In 1939, under Johnson’s supervision, graduate student Mary Tudor conducted a stuttering experiment, using 22 children at an Iowa orphanage. Half received positive reinforcement. But the other half were ridiculed and criticized for their speech, whether or not they actually stuttered. This resulted in a worsening of speech issues for the children who were given negative feedback.

The study was never published due to the multitude of ethical violations. According to The Washington Post , Tudor was remorseful about the damage caused by the experiment and returned to the orphanage to help the children with their speech. Despite his ethical mistakes, the Wendell Johnson Speech and Hearing Clinic at the University of Iowa bears Johnson's name and is a nod to his contributions to the field.

The Dermatologist Who Used Prisoners As Guinea Pigs

One of the biggest breakthroughs in dermatology was the invention of Retin-A, a cream that can treat sun damage, wrinkles, and other skin conditions. Its success led to fortune and fame for co-inventor Albert Kligman, a dermatologist at the University of Pennsylvania . But Kligman is also known for his nefarious dermatology experiments on prisoners that began in 1951 and continued for around 20 years. He conducted his research on behalf of companies including DuPont and Johnson & Johnson.

Kligman’s work often left prisoners with pain and scars as he used them as study subjects in wound healing and exposed them to deodorants, foot powders, and more for chemical and cosmetic companies. Dow once enlisted Kligman to study the effects of dioxin, a chemical in Agent Orange, on 75 inmates at Pennsylvania's Holmesburg Prison. The prisoners were paid a small amount for their participation but were not told about the potential side effects.

In the University of Pennsylvania’s journal, Almanac , Kligman’s obituary focused on his medical advancements, awards, and philanthropy. There was no acknowledgement of his prison experiments. However, it did mention that as a “giant in the field,” he “also experienced his fair share of controversy.”

The Endocrinologist Who Irradiated Prisoners

When the Atomic Energy Commission wanted to know how radiation affected male reproductive function, they looked to endocrinologist Carl Heller . In a study involving Oregon State Penitentiary prisoners between 1963 and 1973, Heller designed a contraption that would radiate their testicles at varying amounts to see what effect it had, particularly on sperm production. The prisoners also were subjected to repeated biopsies and were required to undergo vasectomies once the experiments concluded.

Although study participants were paid, it raised ethical issues about the potential coercive nature of financial compensation to prison populations. The prisoners were informed about the risks of skin burns, but likely were not told about the possibility of significant pain, inflammation, and the small risk of testicular cancer.

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Human Experimentation: An Introduction to the Ethical Issues

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In January 1944, a 17-year-old Navy seaman named Nathan Schnurman volunteered to test protective clothing for the Navy. Following orders, he donned a gas mask and special clothes and was escorted into a 10-foot by 10-foot chamber, which was then locked from the outside. Sulfur mustard and Lewisite, poisonous gasses used in chemical weapons, were released into the chamber and, for one hour each day for five days, the seaman sat in this noxious vapor. On the final day, he became nauseous, his eyes and throat began to burn, and he asked twice to leave the chamber. Both times he was told he needed to remain until the experiment was complete. Ultimately Schnurman collapsed into unconsciousness and went into cardiac arrest. When he awoke, he had painful blisters on most of his body. He was not given any medical treatment and was ordered to never speak about what he experienced under the threat of being tried for treason. For 49 years these experiments were unknown to the public.

The Scandal Unfolds

In 1993, the National Academy of Sciences exposed a series of chemical weapons experiments stretching from 1944 to 1975 which involved 60,000 American GIs. At least 4,000 were used in gas-chamber experiments such as the one described above. In addition, more than 210,000 civilians and GIs were subjected to hundreds of radiation tests from 1945 through 1962.

Testimony delivered to Congress detailed the studies, explaining that “these tests and experiments often involved hazardous substances such as radiation, blister and nerve agents, biological agents, and lysergic acid diethylamide (LSD)....Although some participants suffered immediate acute injuries, and some died, in other cases adverse health problems were not discovered until many years later—often 20 to 30 years or longer.” 1

These examples and others like them—such as the infamous Tuskegee syphilis experiments (1932-72) and the continued testing of unnecessary (and frequently risky) pharmaceuticals on human volunteers—demonstrate the danger in assuming that adequate measures are in place to ensure ethical behavior in research.

Tuskegee Studies

In 1932, the U.S. Public Health Service in conjunction with the Tuskegee Institute began the now notorious “Tuskegee Study of Untreated Syphilis in the Negro Male.” The study purported to learn more about the treatment of syphilis and to justify treatment programs for African Americans. Six hundred African American men, 399 of whom had syphilis, became participants. They were given free medical exams, free meals, and burial insurance as recompense for their participation and were told they would be treated for “bad blood,” a term in use at the time referring to a number of ailments including syphilis, when, in fact, they did not receive proper treatment and were not informed that the study aimed to document the progression of syphilis without treatment. Penicillin was considered the standard treatment by 1947, but this treatment was never offered to the men. Indeed, the researchers took steps to ensure that participants would not receive proper treatment in order to advance the objectives of the study. Although, the study was originally projected to last only 6 months, it continued for 40 years.

Following a front-page New York Times article denouncing the studies in 1972, the Assistant Secretary for Health and Scientific Affairs appointed a committee to investigate the experiment. The committee found the study ethically unjustified and within a month it was ended. The following year, the National Association for the Advancement of Colored People won a $9 million class action suit on behalf of the Tuskegee participants. However, it was not until May 16, 1997, when President Clinton addressed the eight surviving Tuskegee participants and others active in keeping the memory of Tuskegee alive, that a formal apology was issued by the government.

While Tuskegee and the discussed U.S. military experiments stand out in their disregard for the well-being of human subjects, more recent questionable research is usually devoid of obvious malevolent intentions. However, when curiosity is not curbed with compassion, the results can be tragic.

Unnecessary Drugs Mean Unnecessary Experiments

A widespread ethical problem, although one that has not yet received much attention, is raised by the development of new pharmaceuticals. All new drugs are tested on human volunteers. There is, of course, no way subjects can be fully apprised of the risks in advance, as that is what the tests purport to determine. This situation is generally considered acceptable, provided volunteers give “informed” consent. Many of the drugs under development today, however, offer little clinical benefit beyond those available from existing treatments. Many are developed simply to create a patentable variation on an existing drug. It is easy to justify asking informed, consenting individuals to risk limited harm in order to develop new drug therapies for a condition from which they are suffering or for which existing treatments are inadequate. The same may not apply when the drug being tested offers no new benefits to the subjects because they are healthy volunteers, or when the drug offers no significant benefits to anyone because it is essentially a copy of an existing drug.

Manufacturers, of course, hope that animal tests will give an indication of how a given drug will affect humans. However, a full 70 to 75 percent of drugs approved by the Food and Drug Administration for clinical trials based on promising results in animal tests, ultimately prove unsafe or ineffective for humans. 2 Even limited clinical trials cannot reveal the full range of drug risks. A U.S. General Accounting Office (GAO) study reports that of the 198 new drugs which entered the market between 1976 and 1985, 102 (52 percent) caused adverse reactions that premarket tests failed to predict. 3 Even in the brief period between January and August 1997, at least 53 drugs currently on the market were relabeled due to unexpected adverse effects. 4

In the GAO study, no fewer than eight of the drugs in question were benzodiazepines, similar to Valium, Librium, and numerous other sedatives of this class. Two were heterocyclic antidepressants, adding little or nothing to the numerous existing drugs of this type. Several others were variations of cephalosporin antibiotics, antihypertensives, and fertility drugs. These are not needed drugs. The risks taken to develop these drugs by trial participants, and to a certain extent by consumers, were not in the name of science, but in the name of market share.

As physicians, we necessarily have a relationship with the pharmaceutical companies that produce, develop, and market drugs involved in medical treatment. A reflective, perhaps critical posture towards some of the standard practices of these companies—such as the routine development of unnecessary drugs—may help to ensure higher ethical standards in research.

Unnecessary Experimentation on Children

Unnecessary and questionable human experimentation is not limited to pharmaceutical development. In experiments at the National Institutes of Health (NIH), a genetically engineered human growth hormone (hGH) is injected into healthy short children. Consent is obtained from parents and affirmed by the children themselves. The children receive 156 injections each year in the hope of becoming taller.

Growth hormone is clearly indicated for hormone-deficient children who would otherwise remain extremely short. Until the early 1980s, they were the only ones eligible to receive it; because it was harvested from human cadavers, supplies were limited. But genetic engineering changed that, and the hormone can now be manufactured in mass quantities. This has led pharmaceutical houses to eye a huge potential market: healthy children who are simply shorter than average.

Short stature, of course, is not a disease. The problems short children face relate only to how others react to their height and their own feelings about it. The hGH injection, on the other hand, poses significant risks, both physical and psychological.

These injections are linked in some studies to a potential for increased cancer risk, 5-8 are painful, and may aggravate, rather than reduce, the stigma of short stature. 9,10 Moreover, while growth rate is increased in the short term, it is unclear that the final net height of the child is significantly increased by the treatment.

The Physicians Committee for Responsible Medicine worked to halt these experiments and recommended that the biological and psychological effects of hGH treatment be studied in hormone-deficient children who already receive hGH, and that non-pharmacologic interventions to counteract the stigma of short stature also be investigated. Unfortunately, the hGH studies have continued without modification, putting healthy short children at risk.

Use of Placebo in Clinical Research

Whooping cough, also known as pertussis, is a serious threat to infants, with dangerous and sometimes fatal complications. Vaccination has nearly wiped out pertussis in the U.S. Uncertainties remain, however, over the relative merits and safety of traditional whole-cell vaccines versus newer, acellular versions, prompting the NIH to propose an experiment testing various vaccines on children.

The controversial part of the 1993 experiment was the inclusion of a placebo group of more than 500 infants who get no protection at all, an estimated 5 percent of whom were expected to develop whooping cough, compared to the 1.4 percent estimated risk for the study group as a whole. Because of these risks, this study would not be permissible in the U.S. The NIH, however, insisted on the inclusion of a placebo control and therefore initiated the study in Italy where there are fewer restrictions on human research trials. Originally, Italian health officials recoiled from these studies on ethical as well as practical grounds, but persistent pressure from the NIH ensured that the study was conducted with the placebo group.

The use of double-blind placebo-controlled studies is the “gold standard” in the research community, usually for good reason. However, when a well-accepted treatment is available, the use of a placebo control group is not always acceptable and is sometimes unethical. 11 In such cases, it is often appropriate to conduct research using the standard treatment as an active control. The pertussis experiments on Italian children were an example of dogmatic adherence to a research protocol which trumped ethical concerns.

Placebos, Ethics, and Poorer Nations

The ethical problems that placebo-controlled trials raise are especially complicated in research conducted in economically disadvantaged countries. Recently, attention has been brought to studies conducted in Africa on preventing the transmission of HIV from mothers to newborns. Standard treatment for HIV-infected pregnant women in the U.S. is a costly regimen of AZT. This treatment can save the life of one in seven infants born to women with AIDS. 12 Sadly, the cost of AZT treatment is well beyond the means of most of the world’s population. This troubling situation has motivated studies to find a cost-effective treatment that can confer at least some benefit in poorer countries where the current standard of care is no treatment at all. A variety of these studies is now underway in which a control group of HIV-positive pregnant women receives no antiretroviral treatment.

Such studies would clearly be unethical in the U.S. where AZT treatment is the standard of care for all HIV-positive mothers. Peter Lurie, M.D., M.P.H., and Sidney Wolfe, M.D., in an editorial in the New England Journal of Medicine , hold that such use of placebo controls in research trials in poor nations is unethical as well. They contend that, by using placebo control groups, researchers adopt a double standard leading to “an incentive to use as research subjects those with the least access to health care.” 13 Lurie and Wolfe argue that an active control receiving the standard regimen of AZT can and should be compared with promising alternative therapies (such as a reduced dosage of AZT) to develop an effective, affordable treatment for poor countries.

Control Groups and Nutrition

Similar ethical problems are also emerging in nutrition research. In the past, it was ethical for prevention trials in heart disease or other serious conditions to include a control group which received weak nutritional guidelines or no dietary intervention at all. However, that was before diet and lifestyle changes—particularly those using very low fat, vegetarian diets—were shown to reverse existing heart disease, push adult-onset diabetes into remission, significantly lower blood pressure, and reduce the risk of some forms of cancer. Perhaps in the not-too-distant future, such comparison groups will no longer be permissible.

The Ethical Landscape

Ethical issues in human research generally arise in relation to population groups that are vulnerable to abuse. For example, much of the ethically dubious research conducted in poor countries would not occur were the level of medical care not so limited. Similarly, the cruelty of the Tuskegee experiments clearly reflected racial prejudice. The NIH experiments on short children were motivated to counter a fundamentally social problem, the stigma of short stature, with a profitable pharmacologic solution. The unethical military experiments during the Cold War would have been impossible if GIs had had the right to abort assignments or raise complaints. As we address the ethical issues of human experimentation, we often find ourselves traversing complex ethical terrain. Vigilance is most essential when vulnerable populations are involved.

• Frank C. Conahan of the National Security and International Affairs Division of the General Accounting Office, reporting to the Subcommittee of the House Committee on Government Operations.
• Flieger K. Testing drugs in people. U.S. Food and Drug Administration. September 10, 1997.
• U.S. General Accounting Office. FDA Drug Review: Postapproval Risks 1976-85. U.S. General Accounting Office, Washington, D.C., 1990.
• MedWatch, U.S. Food and Drug Administration. Labeling changes related to drug safety. U.S. Food and Drug Administration Home Page; http://www.fda.gov/medwatch/safety.htm . September 10, 1997.
• Arteaga CL, Osborne CK. Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor. Cancer Res . 1989;49:6237-6241.
• Pollak M, Costantino J, Polychronakos C, et al. Effect of tamoxifen on serum insulin-like growth factor I levels in stage I breast cancer patients. J Natl Cancer Inst . 1990;82:1693-1697.
• Stoll BA. Growth hormone and breast cancer. Clin Oncol . 1992;4:4-5.
• Stoll BA. Does extra height justify a higher risk of breast cancer? Ann Oncol . 1992;3:29-30.
• Kusalic M, Fortin C. Growth hormone treatment in hypopituitary dwarfs: longitudinal psychological effects. Canad Psychiatric Asso J . 1975;20:325-331.
• Grew RS, Stabler B, Williams RW, Underwood LE. Facilitating patient understanding in the treatment of growth delay. Clin Pediatr . 1983;22:685-90.
• For a more extensive discussion of the ethical status of placebo-controlled trials see especially: Freedman B, Glass KC, Weijer C. Placebo orthodoxy in clinical research II: ethical, legal and regulatory myths. J Law Med Ethics . 1996;24:252-259.
• Lurie P, Wolfe SM. Unethical trials of interventions to reduce perinatal transmission of the human immunnodeficiency virus in developing countries. N Engl J Med . 1997:337:12:853.

More on Ethical Science

Good Science Digest

A Brief History of Human Challenge Trials

For more than two centuries, scientists have been intentionally infecting patients with dangerous diseases in order to learn more

Theresa Machemer

Correspondent

Physicians promise in the Hippocratic oath to keep their patients from harm, so intentionally exposing people to a deadly disease would seem to run counter to that contract. But with human challenge studies, they do exactly that. In challenge studies, medical professionals purposefully expose patients to illnesses so that they can study the patient’s symptoms and immune system response. Such studies can also help physicians discover what vaccines will work to prevent the affliction. Historically in such experiments, the health of individual patients, usually voluntary but at times, horrifically, not, has been sacrificed for medical knowledge and future treatments.

Researchers are planning new human challenge trials as the race to develop vaccines against Covid-19 is in a full sprint, with Pfizer’s vaccine receiving authorization in several countries and Moderna’s not far behind. But the end of the pandemic won’t just come from these two pharmaceutical breakthroughs. In order to fully contain the spread of Covid-19, many treatments and vaccines may be necessary in order to vaccinate billions of people. And some experts say that the fastest way to test those second-generation vaccines is through human challenge trials.

Imperial College London intends to begin a human challenge study related to Covid-19 as soon as January. During the study, scientists would purposely infect up to 100 young, healthy volunteers with the coronavirus that causes Covid-19 in the hopes of accelerating the search for new vaccines.

Supporters of the controversial Covid-19 human challenge trial argue that if it can be done safely then it provides a uniquely controlled environment to study factors that are difficult to unravel in longer, large-scale Phase III trials of thousands of people. Critics say that challenge studies are either unnecessary because of vaccine successes so far, or should be put on pause until a later date when they can be run safely. Critics also point out that safety is a concern even for young volunteers because scientists do not know how to treat Covid-19 or what its long-term effects are, and evidence presented by the World Health Organization in September showed that at least a fifth of people between 18 and 34 who catch Covid-19 experience prolonged symptoms.

The debate over such a contentious experiment is nothing new. Human challenge trials are as old as inoculation itself. In 1796, English surgeon Edward Jenner tested the world’s first vaccine by exposing his gardener’s 8-year-old son to cowpox and then smallpox. Human challenge trials have since been used to study dozens of diseases from cholera to cancer, but early studies often put participants directly in harm’s way, not always with their knowledge.

Today, challenge studies undergo careful review by boards of experts before they can begin. A key requirement of an ethical study is that volunteers provide informed consent , proving that they understand the risks of joining a study. The first informed consent process was introduced more than a century after Jenner’s human challenge study.

In 1898, as the U.S. warred with Spain in Cuba, yellow fever —which can cause liver damage, nausea, high fever and bleeding—killed 13 times more soldiers than war wounds. So in 1900, the U.S. Army established a commission led by pathologist Walter Reed to figure out how yellow fever spread and how to stop it. Because only humans seemed to fall ill with the disease, Reed and three colleagues on the commission designed a human challenge study to test a leading theory of yellow fever transmission: mosquito bites.

Reed recognized that if he was correct, then the study itself would be incredibly risky. The need to expose volunteers to deadly disease would have to be weighed with the responsibility to keep the volunteers safe.

“The general that created the commission told Walter Reed… that he had to be absolutely sure that no harm would be caused to the volunteers,” says Enrique Chaves-Carballo , a historian of medicine at the University of Kansas. “He was pretty specific about that.”

To balance his superior’s order with the study’s inherent risk, the commission came up with a novel solution: the first informed consent contract. The commission created a document for volunteers to sign, stating that they understood the study’s risks. However, the form suggested that abstaining from the study was risky, too. The contract stated :

“The undersigned understands perfectly well that in the case of the development of yellow fever in him, that he endangers his life to a certain extent but it being entirely impossible for him to avoid the infection during his stay in the island, he prefers to take the chance of contracting it intentionally in the belief that he will receive from the said Commission the greatest care and the most skillful medical service.”

During the experiment, the scientists first allowed mosquitoes to bite yellow fever patients so the insects would pick up the disease. Then, they brought the mosquitoes to healthy volunteers, and allowed the mosquitoes to bite them. When volunteers fell ill, Reed scoured blood samples for the microbe causing their illness.

Those with yellow fever were prescribed complete bed rest and fasting except for “a few sips of champagne” and some pain medication, says Chaves-Carballo. Volunteers received a hefty payment of $100 in gold per mosquito bite, and another $100 if they fell ill.

In the first round of experiments, 11 volunteers got mosquito bites. Two fell ill, and survived. The third man to fall ill, Jesse W. Lazear, was one of the scientists running the study. He was bitten by accident and died of yellow fever 12 days later.

Though Reed considered ending the study after the death of his colleague, the commission instead named a sanitary station Camp Lazear in his honor. And by 1901 , Reed and the commission had shown through their mosquito bite experiments that the insects transmit yellow fever. Inoculation of more volunteers with yellow fever patients’ filtered blood samples showed that a virus causes the disease—making yellow fever the first human virus scientists discovered.

With the disease-causing culprit identified, Reed returned to George Washington University (then Columbian University) to teach, and other scientists picked up the search for a yellow fever vaccine. U.S. army physician William Gorgas and Cuban-born physician Juan Guiteras established an inoculation station for a new round of human challenge studies in Havana. They hoped to learn how to induce light cases of yellow fever with mosquito bites in order to give people immunity. More than 20 volunteers signed up for the first experimental inoculations in 1901, including the only woman to participate in the study, a military nurse named Clara Maass.

Maass was bitten five times without developing yellow fever, and received $100 to send home to her mother and nine siblings in New Jersey—a huge sum compared to her monthly pay of $30 .

Her sixth mosquito bite proved fatal. She and two other volunteers were infected with a particularly violent strain of the virus—the doctors didn’t know how to induce just light cases—and all three died in August of 1901.

“Some of the headlines of the newspapers are like, ‘Nurse Dies for a Hundred Dollars,’” says Chaves-Carballo. “People responded to the fact that she was a young nurse who was trying her best to help her family.”

Public outcry in the U.S. brought the Havana experiments to an end. Maass’ death brought the study’s exorbitant pay under fire, as such a large incentive may have interfered with the participants’ ability to accurately weigh the risk of joining the study. The fact that the study was run by the U.S. Army, and Reed’s participants were members of the military, also brought into question the participants’ ability to freely opt out of the study, says Monica McArthur , pediatrician and infectious disease specialist at the University of Maryland School of Medicine’s Center for Vaccine Development and Global Health.

“In a lot of the studies early on, the Walter Reed experiment and other studies, used what we would now consider vulnerable populations,” people who couldn’t freely agree to participate or make a fully informed decision, says McArthur. “Prisoners, for example, could be enrolled in studies.”

A classic example of a challenge study that relied on a vulnerable population is the Tuskegee Syphilis Study. Beginning in 1932 , the U.S. Public Health Service recruited about 600 poor African American men from around Tuskegee, Alabama, for a study of how syphilis worsens over time. About two-thirds of the men had syphilis, but the study doctors informed them they had “bad blood.”

After receiving this phony diagnosis, the men were persuaded to join the study in exchange for free meals, hospital access and treatment for “bad blood” and other unrelated conditions. The scientists also provided participants a burial stipend that would be paid to their survivors after their deaths.

Only about half of the men with syphilis received a treatment that was usually prescribed in the 1930s: doses of toxic arsenic and mercury. The doctors subjected the participants to blood draws and spinal taps, and after they died of syphilis, autopsies, all in pursuit of more information about the natural course of the disease. The study lasted for decades, and even after the medical community established that penicillin could cure the disease in the 1940s the men did not receive the medication.

In 1972, journalist Jean Heller of the Associated Press brought the Tuskegee Syphilis Study to light and shared how the doctors involved in the study had deceived the men participating. By then, only 74 of the men with syphilis still survived. Public outrage shut the study down three months after the report.

While the Tuskegee Syphilis Study relied on participants who were already ill, other studies exposed otherwise healthy people to deadly diseases. For example, from 1955 to 1970, a pediatrician exposed more than 50 children with mental disabilities to hepatitis in order to identify different strains of the disease and eventually develop vaccines. The trial took place at Willowbrook State School, a home for children and adults with developmental disabilities in Staten Island, New York.

The school was overcrowded and had a lengthy waitlist for new patients. But the study’s principal investigator, Saul Krugman, offered several parents the opportunity to cut the line if they agreed to enroll their children in the study. Krugman told them that their children were likely to catch the disease at the facility anyway, but by joining the study, they would have access to cleaner facilities and a chance at an eventual vaccine.

“I did feel coerced,” said Diana McCourt, who enrolled her daughter in the Willowbrook study, to Forbes ’ Leah Rosenbaum. “I felt like I was denied help unless I took this [opportunity].”

The Willowbrook studies, which ended in 1970, revealed the existence of the A and B strains of hepatitis and sped up the development of a hepatitis B vaccine. But the studies progressed even as some in the medical community criticized Krugman’s methods. In 1966, anesthesiologist Henry K. Beecher published a landmark essay detailing 22 examples of ongoing unethical research on human subjects, including the Willowbrook hepatitis studies, in order to raise awareness and end unethical practices that continued despite the creation of international human experimentation guidelines—the Nuremberg Code in 1947 and the Declaration of Helsinki in 1964.

In addition to the Willowbrook study, Beecher highlighted one study in which melanoma, a serious form of skin cancer, was transferred from a woman to her mother “in the hope of gaining a little better understanding of cancer immunity.” The woman died on the same day that her mother was to receive the melanoma injection, so the doctors knew the cancer was deadly. Her mother died 451 days after receiving the injection.

Beecher concluded that an ethical approach to experimentation requires, first and foremost, the informed consent of study volunteers. “The difficulty of obtaining this is discussed in detail,” he writes, “But it is absolutely essential to strive for it for moral, sociologic and legal reasons. The statement that consent has been obtained has little meaning unless the subject or his guardian is capable of understanding what is to be undertaken and unless all hazards are made clear.”

Human challenge studies became less common after the 1970s with the conclusion of unethical studies that shocked the public. Since then, the Declaration of Helsinki has been amended seven times to clarify ethical standards for human experiments, most recently in October of 2013. The current declaration states that “While the primary purpose of medical research is to generate new knowledge, this goal can never take precedence over the rights and interests of individual research subjects.”

When run well, challenge studies are still uniquely able to provide clear data about infectious diseases. “They are now coming back in favor with very rigorous ethical principles in place,” adds McArthur.

The University of Maryland used human challenge studies in 2012 and 2013 to develop a vaccine for cholera , which was approved by the FDA in 2016. Cholera was an ideal candidate for a safe human challenge study because it is well understood by scientists, is reliably treatable with fluids and antibiotics, and has no long-term effects after the infection is gone.

Informed consent procedures have come a long way since Reed’s contract. Volunteers can ask questions and seek outside guidance, and must pass an assessment designed by the researchers to prove that they understand the risks of a study. And the volunteers have the power to quit. “Every time there’s an encounter with the volunteer, it’s reaffirming that the volunteer is still willing and able to participate,” says McArthur.

According to a statement by Imperial College London, which still needs to have its experimental plan approved by government regulators before researchers can begin recruiting participants, volunteers’ safety is the number one priority. “It would be nice to see exactly how [Imperial College London] explains the risks and benefits to those participating in this study,” says Chaves-Carballo.

Covid-19 is different from other challenge study diseases: Scientists have been studying it for less than a year, physicians have no approved treatments to intervene if a volunteer’s illness becomes severe, and early evidence suggests Covid-19 can cause long-term effects even in young, previously healthy people. The Imperial College London study aims to first identify the minimum dose of coronavirus necessary to cause disease. The study would use that dose of virus to study how vaccines work in the body to prevent Covid-19, to look at potential treatments and study the immune response. The biomedical community remains split on whether such a study should be run, given all of the unknowns around Covid-19.

When scientists develop second- and third-generation vaccines, a challenge study allows researchers to work with just 100 people instead of tens of thousands. That means fewer people are asked to go without the vaccine for the sake of research. And by waiting to conduct a challenge study on Covid-19 until a later date, researchers might get access to new information about risk factors for severe disease, which could help make the study safer.

“I am not a fan of SARS-CoV-2 challenge studies,” says McArthur. “But if I’m playing devil’s advocate against myself, some of the very reasons [not to do a challenge study] that I listed might be reasons that someone else might say that a challenge study is beneficial. Because we don’t know that much about a disease, so we could learn more about it.”

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Theresa Machemer | READ MORE

Theresa Machemer is a freelance writer based in Washington DC. Her work has also appeared in National Geographic and SciShow. Website: tkmach.com

Topic: Human Experimentation

Addressing social justice through the lens of henrietta lacks.

Among the many disruptions of the pandemic, one particular disappointment was the cancellation of the in-person annual meeting of the American Society for Bioethics and Humanities (ASBH), scheduled for Baltimore and set to coincide with the Berman Institute’s 25th Anniversary Celebration and the centennial of Henrietta Lacks’s birth. Yet despite the switch to a virtual format, the Berman Institute was able to host a plenary session that was the talk of the meeting and continues to reverberate.

“Social Justice and Bioethics Through the Lens of the Story of Henrietta Lacks,” was moderated by Jeffrey Kahn and featured Ruth Faden as a panelist. She was joined by Henrietta Lacks’s granddaughter, Jeri Lacks, architect Victor Vines, and Georgetown University Law Center bioethicist Patricia King.

Faden began the session by providing an overview of the Henrietta Lacks story, famed in the context of structural injustice.

“The structural injustice of racism defined in pretty much every way how this story unfolded,” she said. “What is wrong about what happened to the Lacks family engages every core element of human well-being. There were assaults on the social basis of respect, and of self-determination, on attachments, on personal security and on health. Mrs. Lacks and her children were poor Black people in a segregated world in which the most profound injustices of racial oppression were daily features of their lives.”

Faden was followed by Jeri Lacks who expressed the importance of continuing to let the world know about her grandmother’s story.

“Her cells were used to develop the polio vaccine and to treat HIV, and in creating in vitro fertilization. She is a person who continues to give life, and to preserve life,” said Lacks. “No matter what your race, your age, your social circumstances, she continues to improve your life.”

Victor Vines, an architect who was part of the architect team leading programming and planning for the National Museum of African American History and Culture and led the feasibility study for what will be Johns Hopkins University’s Henrietta Lacks Hall, spoke next about addressing racial injustice through architecture and design.

“When we started work on Lacks Hall, we didn’t talk a lot about architecture or design. We talked about what that story is that we want to tell through the building. Meeting with the Lacks family was critically important to that,” Vines said. “We had to understand what they went through and what they care about. The building still has to function and house the Berman Institute, so we had to meet their needs. And we discovered a third client, the East Baltimore community. At the end of the day, this building and university reside within that community, and they will be called to embrace this project – or not.”

King concluded the panel with a riveting and wide-ranging discussion that touched upon intersectionality, segregation, the Tuskegee experiments and participation in clinical trials, COVID, race as a social construct, and the role of consent, all within the framework of Henrietta Lacks’s story.

“Our narratives are important and should be thought of as lessons or homework for institutions,” she said. “They not only document the deep distrust we bring to health encounters but also convey relevant aspects of our lives that should be appreciated.”

As the session ended Kahn noted that perhaps it was fortunate the session had been virtual, so the recording “could be shared with others for posterity. I’m not quite speechless, but maybe close,” he said.

Honoring an Immortal Contribution

Johns Hopkins University President Ronald J. Daniels and Paul B. Rothman, CEO of Johns Hopkins Medicine and dean of the medical faculty of the Johns Hopkins University School of Medicine, along with Berman institute Executive Director Jeffrey Kahn and descendants of Henrietta Lacks, recently announced plans to name a new multidisciplinary building on the Johns Hopkins East Baltimore campus in honor of Henrietta Lacks, who was the source of the HeLa cell line that has been critical to numerous advances in medicine.

Surrounded by descendants of Lacks, Daniels made the announcement at the 9th annual Henrietta Lacks Memorial Lecture in the Turner Auditorium in East Baltimore.

“Through her life and her immortal cells, Henrietta Lacks made an immeasurable impact on science and medicine that has touched countless lives around the world,” Daniels said. “This building will stand as a testament to her transformative impact on scientific discovery and the ethics that must undergird its pursuit. We at Johns Hopkins are profoundly grateful to the Lacks family for their partnership as we continue to learn from Mrs. Lacks’ life and to honor her enduring legacy.”

Henrietta Lacks’ contributions to science were not widely known until the 2010 release of the book The Immortal Life of Henrietta Lacks by Rebecca Skloot, which explored Lacks’ life story, her impact on medical science and important bioethical issues. In 2017, HBO and Harpo Studios released a movie based on the book, with Oprah Winfrey starring as Deborah Lacks, Henrietta Lacks’ daughter.

Several Lacks family members attended today’s event. “It is a proud day for the Lacks family. We have been working with Hopkins for many years now on events and projects that honor our grandmother,” said Jeri Lacks, granddaughter of Henrietta Lacks. “They are all meaningful, but this is the ultimate honor, one befitting of her role in advancing modern medicine.”

The building, which will adjoin the Berman Institute of Bioethics’ current home in Deering Hall will support programs that enhance participation and partnership with members of the community in research that can benefit the community, as well as extend the opportunities to further study and promote research ethics and community engagement in research through an expansion of the Berman Institute and its work.

The story portrayed in The Immortal Life of Henrietta Lacks points to several important bioethical issues, including informed consent, medical records privacy, and communication with tissue donors and research participants.

“The story of Henrietta Lacks has encouraged us all to examine, discuss and wrestle with difficult issues that are at the foundation of the ethics of research, and must inform our relationships with the individuals and communities that are part of that research,” said Jeffrey Kahn, director of the Johns Hopkins University Berman Institute of Bioethics. “As a result, students, faculty and the entire research community at Johns Hopkins and around the world do their work with a greater sensitivity to these critical issues.”

In 2013, Johns Hopkins worked with members of the Lacks family and the National Institutes of Health (NIH) to help broker an agreement that requires scientists to receive permission to use Henrietta Lacks’ genetic blueprint in NIH-funded research.

The NIH committee tasked with overseeing the use of HeLa cells now includes two members of the Lacks family. The medical research community has also made significant strides in improving research practices, in part thanks to the lessons learned from Henrietta Lacks’ story.

“It has been an honor for me to work with the Lacks family on how we can recognize the contribution of Henrietta Lacks to medical research and the community. Their willingness to focus on the positive impact of the HeLa cells has been inspiring to me. The Henrietta Lacks story has led many researchers to rededicate themselves to working more closely with patients,” said Daniel E. Ford, vice dean for clinical investigation in the school of medicine. “The new building will be a hub for the community engagement and collaboration program of the NIH-supported Institute for Clinical and Translational Research.”

Groundbreaking on the building that will be named for Henrietta Lacks is scheduled for 2020 with an anticipated completion in 2022.

To learn more about Henrietta Lacks and the wide-ranging impact of HeLa cells on medical research,

please visit: www.hopkinsmedicine.org/henriettalacks .

Alan Regenberg, MBE

Alan is also engaged in a broad range of research projects and programs, including the Berman Institute’s science programs: the Stem Cell Policy and Ethics (SCOPE) Program ; the Program in Ethics and Brain Sciences (PEBS-Neuroethics) ; and the Hinxton Group , an international consortium on stem cells, ethics and law; and the eSchool+ Initiative . Recent research has focused on using deliberative democracy tools to engage with communities about their values for allocating scarce medical resources like ventilators in disasters like pandemics. Additional recent work has focused on ethical challenges related to gene editing, stem cell research, social media, public engagement, vaccines, and neuroethics. ( Publications )

Joseph Ali, JD

Vaccinating pregnant women against ebola.

In a STAT News opinion piece, Johns Hopkins University experts, including our Ruth Faden, argued it is unfair  to deny pregnant and lactating women the experimental Ebola vaccine if they wish to take it, given the great risk the virus poses to those who are exposed to it.

“From a public health perspective and an ethical perspective, the decision to exclude pregnant and lactating women is utterly indefensible,” they wrote.

The authors are members of Pregnancy Research Ethics for Vaccines, Epidemics, and New Technologies (PREVENT) Working Group, which has brought together an international team of experts in bioethics, maternal immunization, maternal-fetal medicine, obstetrics, pediatrics, philosophy, public health, and vaccine research to provide specific recommendations developed to address this critical gap in vaccine research and development and epidemic response. This group recognizes that excluding pregnant women from efforts to develop and deploy vaccines against emerging threats is not acceptable.

Nancy E. Kass, ScD

Dr. Kass is coeditor (with Ruth Faden) of HIV, AIDS and Childbearing: Public Policy, Private Lives (Oxford University Press, 1996).

She has served as consultant to the President’s Advisory Committee on Human Radiation Experiments, to the National Bioethics Advisory Commission, and to the National Academy of Sciences. Dr. Kass currently serves as the Chair of the NIH Precision Medicine Initiative Central IRB; she previously co-chaired the National Cancer Institute (NCI) Committee to develop Recommendations for Informed Consent Documents for Cancer Clinical Trials and served on the NCI’s central IRB. Current research projects examine improving informed consent in human research, ethical guidance development for Ebola and other infectious outbreaks, and ethics and learning health care. Dr. Kass teaches the Bloomberg School of Public Health’s course on U.S. and International Research Ethics and Integrity, she served as the director of the School’s PhD program in bioethics and health policy from its inception until 2016, and she has directed (with Adnan Hyder) the Johns Hopkins Fogarty African Bioethics Training Program since its inception in 2000. Dr. Kass is an elected member of the Institute of Medicine (now National Academy of Medicine) and an elected Fellow of the Hastings Center.

Jeremy Sugarman, MD, MPH, MA

He was the founding director of the Trent Center for Bioethics, Humanities and History of Medicine at Duke University where he was also a professor of medicine and philosophy. He was appointed as an Academic Icon at the University of Malaya and is a faculty affiliate of the Kennedy Institute of Ethics at Georgetown University.

Dr. Sugarman was the longstanding chair of the Ethics Working Group of the HIV Prevention Trials Network. He is currently a member of the Scientific and Research Advisory Board for the Canadian Blood Service and the Ethics and Public Policy Committees of the International Society for Stem Cell Research. He co-leads the Ethics and Regulatory Core of the NIH Health Care Systems Research Collaboratory and is co-chair of the Johns Hopkins’ Institutional Stem Cell Research Oversight Committee.

Dr. Sugarman has been elected as a member of the American Society of Clinical Investigation, Association of American Physicians, and the National Academy of Medicine (formerly the Institute of Medicine). He is a fellow of the American Association for the Advancement of Science, the American College of Physicians and the Hastings Center. He also received a Doctor of Science, honoris causa, from New York Medical College.

Ugly past of U.S. human experiments uncovered

Shocking as it may seem, U.S. government doctors once thought it was fine to experiment on disabled people and prison inmates. Such experiments included giving hepatitis to mental patients in Connecticut, squirting a pandemic flu virus up the noses of prisoners in Maryland, and injecting cancer cells into chronically ill people at a New York hospital.

Much of this horrific history is 40 to 80 years old, but it is the backdrop for a meeting in Washington this week by a presidential bioethics commission. The meeting was triggered by the government's apology last fall for federal doctors infecting prisoners and mental patients in Guatemala with syphilis 65 years ago.

U.S. officials also acknowledged there had been dozens of similar experiments in the United States — studies that often involved making healthy people sick.

An exhaustive review by The Associated Press of medical journal reports and decades-old press clippings found more than 40 such studies. At best, these were a search for lifesaving treatments; at worst, some amounted to curiosity-satisfying experiments that hurt people but provided no useful results.

Inevitably, they will be compared to the well-known Tuskegee syphilis study. In that episode, U.S. health officials tracked 600 black men in Alabama who already had syphilis but didn't give them adequate treatment even after penicillin became available.

These studies were worse in at least one respect — they violated the concept of "first do no harm," a fundamental medical principle that stretches back centuries.

"When you give somebody a disease — even by the standards of their time — you really cross the key ethical norm of the profession," said Arthur Caplan, director of the University of Pennsylvania's Center for Bioethics.

Attitude similar to Nazi experiments Some of these studies, mostly from the 1940s to the '60s, apparently were never covered by news media. Others were reported at the time, but the focus was on the promise of enduring new cures, while glossing over how test subjects were treated.

Attitudes about medical research were different then. Infectious diseases killed many more people years ago, and doctors worked urgently to invent and test cures. Many prominent researchers felt it was legitimate to experiment on people who did not have full rights in society — people like prisoners, mental patients, poor blacks. It was an attitude in some ways similar to that of Nazi doctors experimenting on Jews.

"There was definitely a sense — that we don't have today — that sacrifice for the nation was important," said Laura Stark, a Wesleyan University assistant professor of science in society, who is writing a book about past federal medical experiments.

The AP review of past research found:

• A federally funded study begun in 1942 injected experimental flu vaccine in male patients at a state insane asylum in Ypsilanti, Mich., then exposed them to flu several months later. It was co-authored by Dr. Jonas Salk, who a decade later would become famous as inventor of the polio vaccine.

Some of the men weren't able to describe their symptoms, raising serious questions about how well they understood what was being done to them. One newspaper account mentioned the test subjects were "senile and debilitated." Then it quickly moved on to the promising results.

• In federally funded studies in the 1940s, noted researcher Dr. W. Paul Havens Jr. exposed men to hepatitis in a series of experiments, including one using patients from mental institutions in Middletown and Norwich, Conn. Havens, a World Health Organization expert on viral diseases, was one of the first scientists to differentiate types of hepatitis and their causes.

A search of various news archives found no mention of the mental patients study, which made eight healthy men ill but broke no new ground in understanding the disease.

• Researchers in the mid-1940s studied the transmission of a deadly stomach bug by having young men swallow unfiltered stool suspension. The study was conducted at the New York State Vocational Institution, a reformatory prison in West Coxsackie. The point was to see how well the disease spread that way as compared to spraying the germs and having test subjects breathe it. Swallowing it was a more effective way to spread the disease, the researchers concluded. The study doesn't explain if the men were rewarded for this awful task.
• A University of Minnesota study in the late 1940s injected 11 public service employee volunteers with malaria, then starved them for five days. Some were also subjected to hard labor, and those men lost an average of 14 pounds. They were treated for malarial fevers with quinine sulfate. One of the authors was Ancel Keys, a noted dietary scientist who developed K-rations for the military and the Mediterranean diet for the public. But a search of various news archives found no mention of the study.
• For a study in 1957, when the Asian flu pandemic was spreading, federal researchers sprayed the virus in the noses of 23 inmates at Patuxent prison in Jessup, Md., to compare their reactions to those of 32 virus-exposed inmates who had been given a new vaccine.
• Government researchers in the 1950s tried to infect about two dozen volunteering prison inmates with gonorrhea using two different methods in an experiment at a federal penitentiary in Atlanta. The bacteria was pumped directly into the urinary tract through the penis, according to their paper.

The men quickly developed the disease, but the researchers noted this method wasn't comparable to how men normally got infected — by having sex with an infected partner. The men were later treated with antibiotics. The study was published in the Journal of the American Medical Association, but there was no mention of it in various news archives.

Though people in the studies were usually described as volunteers, historians and ethicists have questioned how well these people understood what was to be done to them and why, or whether they were coerced.

Victims for science Prisoners have long been victimized for the sake of science. In 1915, the U.S. government's Dr. Joseph Goldberger — today remembered as a public health hero — recruited Mississippi inmates to go on special rations to prove his theory that the painful illness pellagra was caused by a dietary deficiency. (The men were offered pardons for their participation.)

But studies using prisoners were uncommon in the first few decades of the 20th century, and usually performed by researchers considered eccentric even by the standards of the day. One was Dr. L.L. Stanley, resident physician at San Quentin prison in California, who around 1920 attempted to treat older, "devitalized men" by implanting in them testicles from livestock and from recently executed convicts.

Newspapers wrote about Stanley's experiments, but the lack of outrage is striking.

"Enter San Quentin penitentiary in the role of the Fountain of Youth — an institution where the years are made to roll back for men of failing mentality and vitality and where the spring is restored to the step, wit to the brain, vigor to the muscles and ambition to the spirit. All this has been done, is being done ... by a surgeon with a scalpel," began one rosy report published in November 1919 in The Washington Post.

Around the time of World War II, prisoners were enlisted to help the war effort by taking part in studies that could help the troops. For example, a series of malaria studies at Stateville Penitentiary in Illinois and two other prisons was designed to test antimalarial drugs that could help soldiers fighting in the Pacific.

It was at about this time that prosecution of Nazi doctors in 1947 led to the "Nuremberg Code," a set of international rules to protect human test subjects. Many U.S. doctors essentially ignored them, arguing that they applied to Nazi atrocities — not to American medicine.

The late 1940s and 1950s saw huge growth in the U.S. pharmaceutical and health care industries, accompanied by a boom in prisoner experiments funded by both the government and corporations. By the 1960s, at least half the states allowed prisoners to be used as medical guinea pigs.

But two studies in the 1960s proved to be turning points in the public's attitude toward the way test subjects were treated.

The first came to light in 1963. Researchers injected cancer cells into 19 old and debilitated patients at a Jewish Chronic Disease Hospital in the New York borough of Brooklyn to see if their bodies would reject them.

The hospital director said the patients were not told they were being injected with cancer cells because there was no need — the cells were deemed harmless. But the experiment upset a lawyer named William Hyman who sat on the hospital's board of directors. The state investigated, and the hospital ultimately said any such experiments would require the patient's written consent.

At nearby Staten Island, from 1963 to 1966, a controversial medical study was conducted at the Willowbrook State School for children with mental retardation. The children were intentionally given hepatitis orally and by injection to see if they could then be cured with gamma globulin.

Those two studies — along with the Tuskegee experiment revealed in 1972 — proved to be a "holy trinity" that sparked extensive and critical media coverage and public disgust, said Susan Reverby, the Wellesley College historian who first discovered records of the syphilis study in Guatemala.

'My back is on fire!' By the early 1970s, even experiments involving prisoners were considered scandalous. In widely covered congressional hearings in 1973, pharmaceutical industry officials acknowledged they were using prisoners for testing because they were cheaper than chimpanzees.

Holmesburg Prison in Philadelphia made extensive use of inmates for medical experiments. Some of the victims are still around to talk about it. Edward "Yusef" Anthony, featured in a book about the studies, says he agreed to have a layer of skin peeled off his back, which was coated with searing chemicals to test a drug. He did that for money to buy cigarettes in prison.

"I said 'Oh my God, my back is on fire! Take this ... off me!'" Anthony said in an interview with The Associated Press, as he recalled the beginning of weeks of intense itching and agonizing pain.

The government responded with reforms. Among them: The U.S. Bureau of Prisons in the mid-1970s effectively excluded all research by drug companies and other outside agencies within federal prisons.

As the supply of prisoners and mental patients dried up, researchers looked to other countries.

It made sense. Clinical trials could be done more cheaply and with fewer rules. And it was easy to find patients who were taking no medication, a factor that can complicate tests of other drugs.

Additional sets of ethical guidelines have been enacted, and few believe that another Guatemala study could happen today. "It's not that we're out infecting anybody with things," Caplan said.

Still, in the last 15 years, two international studies sparked outrage.

One was likened to Tuskegee. U.S.-funded doctors failed to give the AIDS drug AZT to all the HIV-infected pregnant women in a study in Uganda even though it would have protected their newborns. U.S. health officials argued the study would answer questions about AZT's use in the developing world.

The other study, by Pfizer Inc., gave an antibiotic named Trovan to children with meningitis in Nigeria, although there were doubts about its effectiveness for that disease. Critics blamed the experiment for the deaths of 11 children and the disabling of scores of others. Pfizer settled a lawsuit with Nigerian officials for $75 million but admitted no wrongdoing.

Last year, the U.S. Department of Health and Human Services' inspector general reported that between 40 and 65 percent of clinical studies of federally regulated medical products were done in other countries in 2008, and that proportion probably has grown. The report also noted that U.S. regulators inspected fewer than 1 percent of foreign clinical trial sites.

Monitoring research is complicated, and rules that are too rigid could slow new drug development. But it's often hard to get information on international trials, sometimes because of missing records and a paucity of audits, said Dr. Kevin Schulman, a Duke University professor of medicine who has written on the ethics of international studies.

Syphilis study These issues were still being debated when, last October, the Guatemala study came to light.

In the 1946-48 study, American scientists infected prisoners and patients in a mental hospital in Guatemala with syphilis, apparently to test whether penicillin could prevent some sexually transmitted disease. The study came up with no useful information and was hidden for decades.

Story: U.S. apologizes for Guatemala syphilis experiments

The Guatemala study nauseated ethicists on multiple levels. Beyond infecting patients with a terrible illness, it was clear that people in the study did not understand what was being done to them or were not able to give their consent. Indeed, though it happened at a time when scientists were quick to publish research that showed frank disinterest in the rights of study participants, this study was buried in file drawers.

"It was unusually unethical, even at the time," said Stark, the Wesleyan researcher.

"When the president was briefed on the details of the Guatemalan episode, one of his first questions was whether this sort of thing could still happen today," said Rick Weiss, a spokesman for the White House Office of Science and Technology Policy.

That it occurred overseas was an opening for the Obama administration to have the bioethics panel seek a new evaluation of international medical studies. The president also asked the Institute of Medicine to further probe the Guatemala study, but the IOM relinquished the assignment in November, after reporting its own conflict of interest: In the 1940s, five members of one of the IOM's sister organizations played prominent roles in federal syphilis research and had links to the Guatemala study.

So the bioethics commission gets both tasks. To focus on federally funded international studies, the commission has formed an international panel of about a dozen experts in ethics, science and clinical research. Regarding the look at the Guatemala study, the commission has hired 15 staff investigators and is working with additional historians and other consulting experts.

The panel is to send a report to Obama by September. Any further steps would be up to the administration.

Some experts say that given such a tight deadline, it would be a surprise if the commission produced substantive new information about past studies. "They face a really tough challenge," Caplan said.

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10 Outrageous Experiments Conducted on Humans

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Prisoners, the disabled, the physically and mentally sick, the poor -- these are all groups once considered fair game to use as subjects in your research experiments. And if you didn't want to get permission, you didn't have to, and many doctors and researchers conducted their experiments on people who were unwilling to participate or who were unknowingly participating.

Forty years ago the U.S. Congress changed the rules; informed consent is now required for any government-funded medical study involving human subjects. But before 1974 the ethics involved in using humans in research experiments was a little, let's say, loose. And the exploitation and abuse of human subjects was often alarming. We begin our list with one of the most famous instances of exploitation, a study that eventually helped change the public view about the lack of consent in the name of scientific advancements.

• Tuskegee Syphilis Study
• The Nazi Medical Experiments
• Watson's 'Little Albert' Experiment
• The Monster Study of 1939
• Stateville Penitentiary Malaria Study
• The Aversion Project in South Africa
• Milgram Shock Experiments
• CIA Mind-Control Experiments (Project MK-Ultra)
• The Human Vivisections of Herophilus

10: Tuskegee Syphilis Study

Syphilis was a major public health problem in the 1920s, and in 1928 the Julius Rosenwald Fund, a charity organization, launched a public healthcare project for blacks in the American rural south. Sounds good, right? It was, until the Great Depression rocked the U.S. in 1929 and the project lost its funding. Changes were made to the program; instead of treating health problems in underserved areas, in 1932 poor black men living in Macon County, Alabama, were instead enrolled in a program to treat what they were told was their "bad blood" (a term that, at the time, was used in reference to everything from anemia to fatigue to syphilis). They were given free medical care, as well as food and other amenities such as burial insurance, for participating in the study. But they didn't know it was all a sham. The men in the study weren't told that they were recruited for the program because they were actually suffering from the sexually transmitted disease syphilis, nor were they told they were taking part in a government experiment studying untreated syphilis, the "Tuskegee Study of Untreated Syphilis in the Negro Male." That's right: untreated.

Despite thinking they were receiving medical care, subjects were never actually properly treated for the disease. This went on even after penicillin hit the scene and became the go-to treatment for the infection in 1945, and after Rapid Treatment Centers were established in 1947. Despite concerns raised about the ethics of the Tuskegee Syphilis Study as early as 1936, the study didn't actually end until 1972 after the media reported on the multi-decade experiment and there was subsequent public outrage.

9: The Nazi Medical Experiments

During WWII, the Nazis performed medical experiments on adults and children imprisoned in the Dachau, Auschwitz, Buchenwald and Sachsenhausen concentration camps. The accounts of abuse, mutilation, starvation, and torture reads like a grisly compilation of all nine circles of hell. Prisoners in these death camps were subjected to heinous crimes under the guise of military advancement, medical and pharmaceutical advancement, and racial and population advancement.

Jews were subjected to experiments intended to benefit the military, including hypothermia studies where prisoners were immersed in ice water in an effort to ascertain how long a downed pilot could survive in similar conditions. Some victims were only allowed sea water, a study of how long pilots could survive at sea; these subjects, not surprisingly, died of dehydration. Victims were also exposed to high altitude in decompression chambers -- often followed with brain dissection on the living -- to study high-altitude sickness and how pilots would be affected by atmospheric pressure changes.

Effectively treating war injuries was also a concern for the Nazis, and pharmaceutical testing went on in these camps. Sulfanilamide was tested as a new treatment for war wounds. Victims were inflicted with wounds that were then intentionally infected. Infections and poisonings were also studied on human subjects. Tuberculosis (TB) was injected into prisoners in an effort to better understand how to immunize against the infection. Experiments with poison, to determine how fast subjects would die, were also on the agenda.

The Nazis also performed genetic and racially-motivated sterilizations, artificial inseminations, and also conducted experiments on twins and people of short stature.

8: Watson's 'Little Albert' Experiment

In 1920 John Watson, along with graduate student Rosalie Rayner, conducted an emotional-conditioning experiment on a nine-month-old baby -- whom they nicknamed "Albert B" -- at Johns Hopkins University in an effort to prove their theory that we're all born as blank slates that can be shaped. The child's mother, a wet nurse who worked at the hospital, was paid one dollar for allowing her son to take part.

The "Little Albert" experiment went like this: Researchers first introduced the baby to a small, furry white rat, of which he initially had no fear . (According to reports, he didn't really show much interest at all). Then they re-introduced him to the rat while a loud sound rang out. Over and over, "Albert" was exposed to the rat and startling noises until he became frightened any time he saw any small, furry animal (rats, for sure, but also dogs and monkeys) regardless of noise.

Who exactly "Albert" was remained unknown until 2010, when his identity was revealed to be Douglas Merritte. Merritte, it turns out, wasn't a healthy subject: He showed signs of behavioral and neurological impairment, never learned to talk or walk, and only lived to age six, dying from hydrocephalus (water on the brain). He also suffered from a bacterial meningitis infection he may have acquired accidentally during treatments for his hydrocephalus, or, as some theorize, may have been -- horrifyingly -- intentionally infected as part of another experiment.

In the end, Merritte was never deconditioned, and because he died at such a young age no one knows if he continued to fear small furry things post-experiment.

7: The Monster Study of 1939

Today we understand that stuttering has many possible causes. It may run in some families, an inherited genetic quirk of the language center of the brain. It may also occur because of a brain injury, including stroke or other trauma. Some young children stutter when they're learning to talk, but outgrow the problem. In some rare instances, it may be a side effect of emotional trauma. But you know what it's not caused by? Criticism.

In 1939 Mary Tudor, a graduate student at the University of Iowa, and her faculty advisor, speech expert Wendell Johnson, set out to prove stuttering could be taught through negative reinforcement -- that it's learned behavior. Over four months, 22 orphaned children were told they would be receiving speech therapy, but in reality they became subjects in a stuttering experiment; only about half were actually stutterers, and none received speech therapy.

During the experiment the children were split into four groups:

• Half of the stutterers were given negative feedback.
• The other half of stutterers were given positive feedback.
• Half of the non-stuttering group were all told they were beginning to stutterer and were criticized.
• The other half of non-stutterers were praised.

The only significant impact the experiment had was on that third group; these kids, despite never actually developing a stutter, began to change their behavior, exhibiting low self-esteem and adopting the self-conscious behaviors associated with stutterers. And those who did stutter didn't cease doing so regardless of the feedback they received.

6: Stateville Penitentiary Malaria Study

It's estimated that between 60 to 65 percent of American soldiers stationed in the South Pacific during WWII suffered from a malarial infection at some point during their service. For some units the infection proved to be more deadly than the enemy forces were, so finding an effective treatment was a high priority [source: Army Heritage Center Foundation]. Safe anti-malarial drugs were seen as essential to winning the war.

Beginning in 1944 and spanning over the course of two years, more than 400 prisoners at the Stateville Penitentiary in Illinois were subjects in an experiment aimed at finding an effective drug against malaria . Prisoners taking part in the experiment were infected with malaria, and then treated with experimental anti-malarial treatments. The experiment didn't have a hidden agenda, and its unethical methodology didn't seem to bother the American public, who were united in winning WWII and eager to bring the troops home — safe and healthy. The intent of the experiments wasn't hidden from the subjects, who were at the time praised for their patriotism and in many instances given shorter prison sentences in return for their participation.

5: The Aversion Project in South Africa

If you were living during the apartheid era in South Africa, you lived under state-regulated racial segregation. If that itself wasn't difficult enough, the state also controlled your sexuality.

The South African government upheld strict anti-homosexual laws. If you were gay you were considered a deviant — and your homosexuality was also considered a disease that could be treated. Even after homosexuality ceased to be considered a mental illness and aversion therapy as a way to cure it debunked, psychiatrists and Army medical professionals in the South African Defense Force (SADF) continued to believe the outdated theories and treatments. In particular, aversion therapy techniques were used on prisoners and on South Africans who were forced to join the military under the conscription laws of the time.

At Ward 22 at 1 Military hospital in Voortrekkerhoogte, Pretoria, between 1969 and 1987 attempts were made to "cure" perceived deviants. Homosexuals, gay men and lesbians were drugged and subjected to electroconvulsive behavior therapy while shown aversion stimuli (same-sex erotic photos), followed by erotic photos of the opposite sex after the electric shock. When the technique didn't work (and it absolutely didn't), victims were then treated with hormone therapy, which in some cases included chemical castration. In addition, an estimated 900 men and women also underwent gender reassignment surgery when subsequent efforts to "reorient" them failed — most without consent, and some left unfinished [source: Kaplan ].

4: Milgram Shock Experiments

Ghostbuster Peter Venkman, who is seen in the fictional film conducting ESP/electro-shock experiments on college students, was likely inspired by social psychologist Stanley Milgram's famous series of shock experiments conducted in the early 1960s. During Milgram's experiments "teachers" — Americans recruited for a Yale study they thought was about memory and learning — were told to read lists of words to "learners" (actors, although the teachers didn't know that). Each person in the teacher role was instructed to press a lever that would deliver a shock to their "learner" every time he made a mistake on word-matching quizzes. Teachers believed the voltage of shocks increased with each mistake, and ranged from 15 to 450 possible volts; roughly two-thirds of teachers shocked learners to the highest voltage , continuing to deliver jolts at the instruction of the experimenter.

In reality, this wasn't an experiment about memory and learning; rather, it was about how obedient we are to authority. No shocks were actually given.

Today, Milgram's shock experiments continue to be controversial; while they're criticized for their lack of realism, others point to the results as important to how humans behave when under duress. In 2010 the results of Milgram's study were repeated — with about 70 percent of teachers obediently administering what they believed to be the highest voltage shocks to their learners.

3: CIA Mind-Control Experiments (Project MK-Ultra)

If you're familiar with "Men Who Stare at Goats" or "The Manchurian Candidate" then you know: There was a period in the CIA's history when they performed covert mind-control experiments. If you thought it was fiction, it wasn't.

During the Cold War the CIA started researching ways they could turn Americans into CIA-controlled "superagents," people who could carry out assassinations and who wouldn't be affected by enemy interrogations. Under what was known as the MK-ULTRA project, CIA researchers experimented on unsuspecting American (and Canadian) citizens by slipping them psychedelic drugs, including LSD , PCP and barbiturates, as well as additional — and additionally illegal — methods such as hypnosis, and, possibly, chemical, biological, and radiological agents. Universities participated, mostly as a delivery system, also without their knowledge. The U.S. Department of Veterans Affairs estimates 7,000 soldiers were also involved in the research, without their consent.

The project endured for more than 20 years, during which the agency spent about $20 million. There was one death tied to the project, although more were suspected; tin 1973 the CIA destroyed what records were kept.

2: Unit 731

Using biological warfare was banned by the Geneva Protocol in 1925, but Japan rejected the ban. If germ warfare was effective enough to be banned, it must work, military leaders believed. Unit 731 , a secret unit in a secret facility — publicly known as the Epidemic Prevention and Water Supply Unit — was established in Japanese-controlled Manchuria, where by the mid-1930s Japan began experimenting with pathogenic and chemical warfare and testing on human subjects. There, military physicians and officers intentionally exposed victims to infectious diseases including anthrax , bubonic plague, cholera, syphilis, typhus and other pathogens, in an effort to understand how they affected the body and how they could be used in bombs and attacks in WWII.

In addition to working with pathogens, Unit 731 conducted experiments on people, including — but certainly not limited to — dissections and vivisections on living humans, all without anesthesia (the experimenters believed using it would skew the results of the research).

Many of the subjects were Chinese civilians and prisoners of war, but also included Russian and American victims among others — basically, anyone who wasn't Japanese was a potential subject. Today it's estimated that about 100,000 people were victims within the facility, but when you include the germ warfare field experiments (such as reports of Japanese planes dropping plague-infected fleas over Chinese villages and poisoning wells with cholera) the death toll climbs to estimates closer to 250,000, maybe more.

Believe it or not, after WWII the U.S. granted immunity to those involved in these war crimes committed at Unit 731 as part of an information exchange agreement — and until the 1980s, the Japanese government refused to admit any of this even happened.

1: The Human Vivisections of Herophilus

Ancient physician Herophilus is considered the father of anatomy. And while he made significant discoveries during his practice, it's how he learned about internal workings of the human body that lands him on this list.

Herophilus practiced medicine in Alexandria, Egypt, and during the reign of the first two Ptolemaio Pharoahs was allowed, at least for about 30 to 40 years, to dissect human bodies, which he did, publicly, along with contemporary Greek physician and anatomist Erasistratus. Under Ptolemy I and Ptolemy II, criminals could be sentenced to dissection and vivisection as punishment, and it's said the father of anatomy not only dissected the dead but also performed vivisection on an estimated 600 living prisoners [source: Elhadi ].

Herophilus made great strides in the study of human anatomy — especially the brain , eyes, liver, circulatory system, nervous system and reproductive system, during a time in history when dissecting human cadavers was considered an act of desecration of the body (there were no autopsies conducted on the dead, although mummification was popular in Egypt at the time). And, like today, performing vivisection on living bodies was considered butchery.

Frequently Asked Questions

How have these experiments influenced current ethical standards in research, what protections are in place today to prevent similar unethical research on humans, lots more information, author's note.

There is no denying that involving living, breathing humans in medical studies have produced some invaluable results, but there's that one medical saying most of us know, even if we're not in a medical field: first do no harm (or, if you're fancy, primum non nocere).

Related Articles

• What will medicine consider unethical in 100 years?
• How Human Experimentation Works
• Top 5 Crazy Government Experiments
• 10 Cover-ups That Just Made Things Worse
• 10 Really Smart People Who Did Really Dumb Things
• How Scientific Peer Review Works

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Science-Based Medicine

Exploring issues and controversies in the relationship between science and medicine

Ethics in human experimentation in science-based medicine

Science-based medicine depends upon human experimentation. Scientists can do the most fantastic translational research in the world, starting with elegant hypotheses, tested through in vitro and biochemical experiments, after which they are tested in animals. They can understand disease mechanisms to the individual amino acid level in a protein or nucleotide in a DNA molecule. However, without human testing, they will never know if the end results of all that elegant science will actually do what it is intended to do and to make real human patients better. They will never know if the fruits of all that labor will actually cure disease. However, it is in human experimentation where the ethics of science most tend to clash with the mechanisms of science. We refer to “science-based medicine” ( SBM ) as “based” in science, but not science, largely because medicine can never be pure science. Science has resulted in amazing medical advances over the last century, but if there is one thing that we have learned it’s that, because clinical trials involve living, breathing, fellow human beings, what is the most scientifically rigorous trial design might not be the most ethical.

About a week ago, the AP reported that experiments and clinical trials that resemble the infamous Tuskegee syphilis study and the less well known, but recently revealed Guatemala syphilis experiment were far more common than we might like to admit. As I sat through talks about clinical trial results at the Society of Surgical Oncology meeting in San Antonio over the weekend, the revelations of the last week reminded me that the intersection between science and ethics in medicine can frequently be a very tough question indeed. In fact, in many of the discussions, questions of what could or could not be done based on ethics were frequently mentioned, such as whether it is ethically acceptable or possible to do certain followup trials to famous breast cancer clinical trials. Unfortunately, it was not so long ago that such questions were answered in ways that bring shame on the medical profession.

More than Tuskegee and Guatemala

The most notorious of highly unethical human experiments outside of Nazi Germany and the Japanese empire during World War II is the infamous Tuskegee syphilis study . This study, conducted by our very own Public Health Service (PHS) was conducted between 1932 and 1972 and examined the natural progression of untreated syphilis in poor black men who received free health care from the government. In 1932, when this study was conceived it was not inherently unethical. At the time there were precious few treatments for syphilis, and none of them worked very well. Consequently, observing the progression of syphilis, using the treatments available at the time, and following the subjects prospectively constituted a reasonable trial design. However, in the late 1930s and early 1940s, penicillin became available, and by 1947 was the standard of care for treating syphilis. When campaigns to eradicate syphilis came to the county in which most of the subjects, study researchers prevented their subjects from participating. In essence, even after an effective treatment for syphilis had become widely available, study still researchers denied it to their subjects. By the end of the study in 1972, of the original 399 men in the study, 28 had died of syphilis; 100 were dead of related complications; 40 wives had been infected with syphilis; and 19 children had been born with congenital syphilis. The rationale for not providing effective treatment for these men and even discouraging them from undergoing such treatment? This :

Such individuals seemed to offer an unusual opportunity to study the untreated syphilitic patients from the beginning of the disease to the death of the infected person. An opportunity was also offered to compare the syphilitc process uninfluenced by modern treatment, with the results attained when treatment had been given.

Worse, there was no informed consent, and considerable inducements were offered to the men to join the study.

The Tuskegee syphilis study, unfortunately, is not the only abuse committed by the PHS. About six months ago, it was revealed that these sorts of experiments had been more widespread than commonly believed. Indeed, in the 1940s in Guatemala, the PHS had gone one better in that they had deliberately infected prison inmates in Guatemala with syphilis. As I described in a lot more detail when the revelations first hit the press, prostitutes who had tested positive for syphilis were recruited to visit the men in prison. The hypothesis to be tested was whether prophylactic penicillin treatment could prevent infection, and the other purpose was to define the response of syphilis to penicillin treatment. Again, there was no real informed consent. Worse, subjects were intentionally infected with a potentially fatal disease. True, they were treated, but treatment is not 100% effective, and one has to wonder if the prisoners, a vulnerable population, understood the nature of the risks they were being induced to take.

On Sunday, AP medical writer Michael Stobbe published a long article detailing the sordid history of medical research in the U.S. before the 1970s. His timing was not coincidental, because on Tuesday in Washington, DC, there was a meeting of a presidential bioethics committee, the Commission for the Study of Bioethical Issues, triggered by the revelations last fall about the Guatemala syphilis experiment 65 years ago. Those revelations led the AP to do an exhaustive review of reports from medical journals and press clippings, and the AP found at least 40 studies similar to the Guatemala syphilis study in that patients were put at risk for serious disease or, even worse, healthy people were intentionally made ill to study disease. Some of these abuses are well known, others much less so.

Here are some examples from the AP article:

The AP review of past research found: A federally funded study begun in 1942 injected experimental flu vaccine in male patients at a state insane asylum in Ypsilanti, Mich., then exposed them to flu several months later. It was co-authored by Dr. Jonas Salk, who a decade later would become famous as inventor of the polio vaccine. Some of the men weren’t able to describe their symptoms, raising serious questions about how well they understood what was being done to them. One newspaper account mentioned the test subjects were “senile and debilitated.” Then it quickly moved on to the promising results. In federally funded studies in the 1940s, noted researcher Dr. W. Paul Havens Jr. exposed men to hepatitis in a series of experiments, including one using patients from mental institutions in Middletown and Norwich, Conn. Havens, a World Health Organization expert on viral diseases, was one of the first scientists to differentiate types of hepatitis and their causes. A search of various news archives found no mention of the mental patients study, which made eight healthy men ill but broke no new ground in understanding the disease. Researchers in the mid-1940s studied the transmission of a deadly stomach bug by having young men swallow unfiltered stool suspension. The study was conducted at the New York State Vocational Institution, a reformatory prison in West Coxsackie. The point was to see how well the disease spread that way as compared to spraying the germs and having test subjects breathe it. Swallowing it was a more effective way to spread the disease, the researchers concluded. The study doesn’t explain if the men were rewarded for this awful task. A University of Minnesota study in the late 1940s injected 11 public service employee volunteers with malaria, then starved them for five days. Some were also subjected to hard labor, and those men lost an average of 14 pounds. They were treated for malarial fevers with quinine sulfate. One of the authors was Ancel Keys, a noted dietary scientist who developed K-rations for the military and the Mediterranean diet for the public. But a search of various news archives found no mention of the study. For a study in 1957, when the Asian flu pandemic was spreading, federal researchers sprayed the virus in the noses of 23 inmates at Patuxent prison in Jessup, Md., to compare their reactions to those of 32 virus-exposed inmates who had been given a new vaccine. Government researchers in the 1950s tried to infect about two dozen volunteering prison inmates with gonorrhea using two different methods in an experiment at a federal penitentiary in Atlanta. The bacteria was pumped directly into the urinary tract through the penis, according to their paper. The men quickly developed the disease, but the researchers noted this method wasn’t comparable to how men normally got infected — by having sex with an infected partner. The men were later treated with antibiotics. The study was published in the Journal of the American Medical Association , but there was no mention of it in various news archives.

Stobbe goes on to point out the “Holy Trinity” of news stories in the 1960s and early 1970s that brought to light the sorts of activities that we now consider abuses in medical research. The last of these was, of course, the Tuskegee syphilis study. The first of these occurred in 1963, when it came to light that researchers had injected cancer cells into elderly debilitated patients at the Jewish Chronic Disease Hospital in Brooklyn to discover whether their bodies would reject them. With our knowledge of tumor immunology now, we can look back on this experiment and know that the odds of any harm were quite small because tumors, with very, very rare exceptions, are not transplantable in humans. Our bodies recognize cells from another person to be foreign, whether they are cancer or not, and quickly destroy them. However, at the time, based on what was known, undoubtedly the scientists thought that there was at least a chance that these tumor cells would form cancers in the patients into whom they were injected, the denial of the hospital director who deemed the cells “harmless,” notwithstanding. (Indeed, the hospital director strikes me as either lying or deluded.) Otherwise, why seek to answer the question? Just as bad, there was no informed consent, the justification being that the cells were thought to be “harmless.” More details can be found here . The outcome was that the Board of Regents censured the researchers and suspended the licenses of two of the doctors involved. Later, however, they stayed the suspensions and instead put the doctors on probation for one year. There were no repercussions for the hospital or for Memorial Sloan-Kettering Cancer Center, where one of the investigators was on faculty.

The third of the “Holy Trinity” was an infamous experiment in Staten Island at the Willowbrook State School, which was a school for children with mental retardation. During the mid-1960s, children there were intentionally infected with hepatitis in order to determine whether gamma globulin could cure it. Besides the targeting of a vulnerable population (children and teens with profound mental retardation), this study demonstrated a number of problematic issues as well . First, the investigators rationalized infecting these children by rationalizing that hepatitis was so endemic in the facility due to the fact that most of the children there were incapable of being toilet trained that over 70% of new residents became infected within a year. This, of course, leads to the obvious question , namely: If that were the case then why not study the effect of gamma globulin on children who were infected normally? More disturbing, again investigators played fast and loose with informed consent, the form being worded in a vague and ambiguous manner that played down the fact that the children were going to be intentionally infected with hepatitis and implying that the serum they would be given would be an experimental vaccine. Finally, as is the case in many such studies, there was an element of coercion. Willowbrook at the time was very crowded, with long waiting lists for children to be admitted. At times, there was only room in the experimental wing. For parents who could not afford to take care of their children, this situation could bring considerable pressure to bear to “persuade” them to “do the right thing.”

Changing ethics

In studying the history of medicine and clinical trials, what never ceases to amaze me is the different attitudes that physicians and scientists had towards their human subjects not all that long ago. Remember, it was primarily in the 1960s and 1970s when attitudes began to change. Before the 1970s, for instance, researchers thought little of using prisoners for experiments, even though prisoners are correctly considered a population that is vulnerable and for whom true informed consent without coercion is difficult to obtain without special attention to making it happen. Indeed, in his news story Stobbe recounts an anecdote of a man at Holmesburg Prison in Philadelphia who agreed in exchange for cigarette money to have the skin peeled off of his back and searing chemicals painted on the open wounds in order to test a drug. Similarly, as the Willowbrook story shows us, it was not really all that long ago when scientists apparently felt justified in infecting profoundly mentally retarded children with hepatitis on the basis of at best dubious ethical justification.

Arguably, this willingness to experiment on children who were not normal and who were never going to be able to contribute to society was a holdover from the eugenics movement earlier in the 20th century. It’s important to remember that, however much eugenics was discredited by the Nazis, prior to the Holocaust Hitler was actually quite the admirer of American eugenics policies, drawing inspiration from them. Another factor that is frequently invoked as an explanation for the willingness of American scientists to flout ethical considerations is war, particularly World War II and then the Cold War, the resulting idea that it was “us against them,” and that for us to win would require shared sacrifice in the name of the nation. After all, the scientific primacy of the U.S. was viewed as one of the most critical sources of our economic and military strength. Moreover, the concept of the Cold War could be generalized to other “wars,” such as the “war on disease” or the current “war on cancer” that I’ve written about twice in the last month. As Stobbe put it:

Attitudes about medical research were different then. Infectious diseases killed many more people years ago, and doctors worked urgently to invent and test cures. Many prominent researchers felt it was legitimate to experiment on people who did not have full rights in society — people like prisoners, mental patients, poor blacks. It was an attitude in some ways similar to that of Nazi doctors experimenting on Jews. “There was definitely a sense — that we don’t have today — that sacrifice for the nation was important,” said Laura Stark, a Wesleyan University assistant professor of science in society, who is writing a book about past federal medical experiments.

There was clearly also more than a little hubris at play as well:

It was at about this time that prosecution of Nazi doctors in 1947 led to the “Nuremberg Code,” a set of international rules to protect human test subjects. Many U.S. doctors essentially ignored them, arguing that they applied to Nazi atrocities — not to American medicine.

Finally, with the rise of large pharmaceutical companies in the 1940s and 1950s, increasingly there was more of a profit motive than a purely scientific one. Drugs needed to be tested, and prisoners provided a convenient source of young, healthy men upon which to test new products. Hubris, profit, and a wartime attitude that sacrificing for the good of the nation all swirled together into a mixture toxic to medical ethics during World War II and well into the postwar period. In having defeated the Nazis, we failed to learn a lesson from what had happened in Germany, where one of the most technologically and medically advanced societies then on the face of the earth did horrible things in the name of its ideology.

Yes, it is true that American scientists did not intentionally expose prisoners to freezing water in experiments designed to find better ways of rewarming pilots shot down over frigid waters or sailors who survived the sinking of their ship, as Nazi doctors did. It is also true that American scientists did not intentionally irradiate men’s testicles and women’s ovaries in order to develop a means of rapid sterilization, causing horrific bowel and bladder complications, especially in women, as Nazi scientists did, although American scientists did subject many to various radioactive substances in the name of research. Nor did American scientists inject dyes into the eyes of children in order to try to turn them blue, as Dr. Mengele did. On the other hand, American scientists did, as we have seen, intentionally infect prisoners and mentally retarded children (the same sort of children that the Nazis would have called “life unworthy of life”) with diseases and then treat them, just as Nazi physicians intentionally infected concentration camp inmates with various diseases in order to determine the efficacy of different treatments or as Japanese physicians did when they intentionally broke the limbs of prisoners and contaminated them with bacteria-laden dirt. American offenses were different in scale and horror, but not significantly different in kind. Unfortunately, it was not until the 1970s, years after the international Helsinki Declaration was first published, until the Belmont Report was adopted and then not until the 1990s when The Common Rule became the basis of all federal regulations protecting human research subjects, as I have described before .

Could it happen again?

Fortunately, as one who now participates in clinical trials and clinical trial development, given the current level of regulation on human subjects research by the federal government, I have a hard time imagining how abuses such as the one’s I’ve described could happen again now. The amount of paperwork, regulation, and oversight of clinical trials has become so burdensome and complex that sometimes I wonder why I or anyone else would want to continue doing clinical research. Unfortunately, Stobbe doesn’t sound too optimistic. Actually, it’s not so much Stobbe, but rather the presidential Commission for the Study of Bioethical Issues, as Stobbe documents in a followup story :

Speakers noted that over the last several decades, as many as 1,000 rules, regulations and guidelines have been enacted worldwide to ensure the ethical conduct of medical research. In the United States, there are rules to protect people in every study done by federal scientists, funded by federal agencies or those testing a product requiring federal approval to be sold. But that oversight is inconsistent — ethical rules can vary among federal agencies. What’s more, if federal funding or review is not involved, an unethical study could be done and no one in authority would ever know about it. “We have a leaky system,” said Eric Meslin, director of the Indiana University Center for Bioethics. Dr. Robert Califf, Duke University’s vice chancellor for clinical research, agreed there are weaknesses. “It’s night and day and what you could do in the ‘good old days’ with no one knowing about it. But there’s no 100 percent guarantee. There still will be bad things that will happen,” he said.

In terms of pharmaceutical companies, there are clearly loopholes when it comes to overseas studies. Indeed, pharmaceutical companies have been doing more and more studies overseas. Although federal law states that such studies, if they are funded by the federal government or if they are to be used as part of an application for FDA approval of a drug, that is not always enough of a guarantee of oversight :

Last year, the U.S. Department of Health and Human Services’ inspector general reported that between 40 and 65 percent of clinical studies of federally regulated medical products were done in other countries in 2008, and that proportion probably has grown. The report also noted that U.S. regulators inspected fewer than 1 percent of foreign clinical trial sites.

Clearly, this is an unacceptable level of oversight, particularly outside of developed countries, such as those in Europe, where clinical trial oversight is comparable to that in the U.S.

Ironically, two examples come to mind of clinical trials that show the holes in our regulatory system for human subjects protection, both of which I have written about right here on SBM before. The first trial was a trial of homeopathic remedies for infants with infectious diarrhea in Honduras, as I wrote about here and Wally Sampson wrote about here . At the time I couldn’t figure out how the investigators at the University of Washington managed to get this study through their IRB, but somehow they did, demonstrating that an IRB is not a guarantee against the approval of totally unethical and scientifically worthless experiments. Fortunately, as far as I can tell, no infant was injured, but the potential was definitely there. Then, let’s not forget the Gonzalez trial, a trial of a regimen of what can best be described as pure quackery consisting of up to 150 supplement pills a day, various nutritional pseudoscience, and daily (or more) coffee enemas. The results were devastating , in that subjects on the standard-of-care chemotherapy arm lived three times longer than those on the Gonzalez protocol arm. Such is the effect of the National Center for Complementary and Alternative Medicine ( NCCAM ) on research ethics.

The more disturbing example is Mark and David Geier, the father-son tag team of anti-vaccine activists who fervently believe that mercury in vaccines causes autism and somehow came up with an idea that can only be described as dangerously wacky, namely that by suppressing testosterone with a powerful drug (Lupron) they could make the quackery known as chelation therapy “work better” at chelating mercury from the brains of autistic children. The reason? Because “testosterone sheets” bind mercury and keep it from being chelated! In pursuing this research, the Geiers have created an IRB stocked with their cronies and fellow anti-vaccinationists to “oversee” the research, as Kathleen Seidel has so thoroughly documented . In the process, autistic children were subjected to a powerful drug that depresses their sex hormone levels, which is why its use is often referred to as “chemical castration.” Predictably, Anne Dachel, Media Editor over at the anti-vaccine crank blog Age of Autism, has leapt all over this story as “evidence” that vaccines must be dangerous and that unethical scientists have been lying all along about the science showing tthat there is no evidence that vaccines cause autism:

Either Stobbe is a naïve and trusting soul and can’t consider that the same government that allowed horrific medical experiments in the past also allowed our children to become vaccine guinea pigs, or he’s afraid of an issue that’s just too controversial to talk about here and now. It’s much safer to attack what went on in the last century. Maybe 70 years from now, some enterprising reporter will bring up the ethics of injecting known neurotoxins in pregnant women, babies, and small children. Maybe around 2080, they’ll ask why no one ever demanded independent studies on the cumulative effect of so many vaccines, so soon, on the health of a baby. Or why there was never a simple vax-nonvax comparison study looking at autism rates.

Or maybe in 2050, Dachel will understand that this is the sort of work that’s been replicated so many times and done in so many different countries that even if you were to throw out all the U.S. data it wouldn’t change the conclusion that vaccines do not cause autism. She also overlooks the fact that the vast majority of the studies that have failed to find a link between vaccines and autism were performed after the adoption of the Common Rule and much-increased federal oversight over clinical trials. Of course, stories like Stobbe’s make it easier for cranks to attack the entire U.S. clinical research enterprise as corrupt and unethical. However, that is not the reason why we need to close the loopholes in our current clinical trial regulations. We need to do it because it is the right thing to do.

We at SBM argue that medicine should be based on science, rather than be a science, because we realize that medicine can never be completely scientific. There are too many human variables, not the least of which are patient values, individual patient situations, and resources. Another reason is the clinical trial process itself. Sometimes the most scientifically rigorous clinical trial design is not the most ethical design; indeed, sometimes it might be downright unethical. One example is, as I have pointed out , the aforementioned study of vaccinated versus unvaccinated children that seems to be every anti-vaccine activist’s most fervent dream. The most scientifically rigorous design for such a study would be a randomized, double-blind, placebo-controlled trial. However, such a trial would leave half of its participants completely unprotected against potentially deadly childhood infectious diseases, making it totally unethical to perform, even if it could be scientifically and fiscally justified based on existing preliminary data, which it really cannot.

Perhaps a better example is how placebo-controlled trials have almost gone the way of the dodo in cancer chemotherapy trials. Most oncology trials are now designed to test a new drug against the current standard of care or the new drug plus the standard of care versus standard of care alone. This is because our ethical considerations have evolved such that we now no longer consider giving placebos to cancer patients to be ethical unless there truly is no existing effective treatment for their cancer or if we truly do not know if the proposed treatment is better than observation alone and observation alone is currently the standard of care. As I have described before, in clinical trials, there must be clinical equipoise ; i.e., based on the scientific evidence as it is known at the time the trial begins, a reasonable scientific assessment of the risks and benefits must conclude that the risks to the experimental group are either minimal or outweighed by the potential benefits. Here’s another thought to chew on. Experiments in which people were intentionally exposed to infectious agents and then subjected to various treatments to cure the disease thus caused are potentially the most scientifically rigorous way of all to test such treatments in humans because they allow control of the start of the infection, the amount of bacteria injected, and many other variables that can’t be so easily controlled in “wild” cases of infectious disease. However, because such experiments violate the precept of, “First, do no harm,” they are utterly unethical and now properly condemned by any physician with a shred of ethics. That we should require laws, rules, and regulations to prevent such unethical experiments by scientists is unfortunately, but scientists are no different than any other person. Not all of them are ethical; some are completely unethical. Some can be corrupted.

There will always be unethical scientists, at least as long as there are unethical people. That’s why we need laws to protect human subjects. However, we must also remember that the protection of human subjects is a balancing act. Go too far in the direction of lax regulation, and incidents such as those described in Stobbe’s article will start to happen again. Go too far in the other direction, and the pace of discovery will grind to a halt. Key to finding the balance is to respect patient autonomy and to provide true informed consent that accurately balances risks versus benefits and to protect patients from any form of coercion. Doing so without making the clinical trial process so onerous that researchers flee the field while at the same time protecting patients from foreseeable harms will be the challenge.

Dr. Gorski's full information can be found here , along with information for patients. David H. Gorski, MD, PhD, FACS is a surgical oncologist at the Barbara Ann Karmanos Cancer Institute specializing in breast cancer surgery, where he also serves as the American College of Surgeons Committee on Cancer Liaison Physician as well as an Associate Professor of Surgery and member of the faculty of the Graduate Program in Cancer Biology at Wayne State University. If you are a potential patient and found this page through a Google search, please check out Dr. Gorski's biographical information, disclaimers regarding his writings, and notice to patients here .

• Posted in: Clinical Trials , Medical Ethics , Pharmaceuticals , Science and the Media

Posted by David Gorski

• American History

The 10 Cruelest Human Experimentation Cases in History

“First, do no harm,” is the oath taken by physicians the world over. And this has been the case for centuries now. For the most part, these men and women of science stay faithful to this oath, even defying orders to the contrary. But sometimes they not only break it, they do so in the worst way imaginable. There have been numerous instances of doctors and other scientists going way beyond the limitations of what’s moral or ethical in the name of ‘progress’. They have used humans as experimental guinea pigs for their tests.

In many cases, the test subjects were either kept in ignorance about what an experiment involved or they were simply in no position to offer their resistance or consent. Of course, it may well be the case that such dubious methods produced results. Indeed, some of the most controversial experiments of the past century produced results that continue to inform scientific understanding to this day. But that will never mean such experiments will be seen as just. Sometimes, the perpetrators of cruel research lose their good names or reputations. Sometimes they are prosecuted for their attempts to ‘play God’. Or sometimes they just get away with it.

You might want to brace yourself as we look at the ten weirdest and cruelest human experiments carried out in history:

Dr. Shiro Ishii and Unit 731

During World War II, Imperial Japan committed a number of crimes against humanity. But perhaps few were crueler than the experiments that were conducted at Unit 731. Part of the Imperial Japanese Army, this was a super-secret unit dedicated to undertaking research into biological and chemical weapons. Quite simply, the Imperial authority wanted to build weapons that were deadlier – or just crueler – than anything that had gone before. And they weren’t opposed to using human guinea pigs to test their creations.

Based in Harbon, the biggest city of Manchuko, the part of north-east China that Japan made its puppet state, Unit 731 was constructed between 1934 and 1939. Overseeing its construction was General Shiro Ishii. Though he was a medical doctor, Ishii was also a fanatical soldier and so he was happy to set his ethics aside in the name of total victory for Imperial Japan. In all, it’s estimated that as many as 3,000 men, women and children were used as forced participants in the experiments conducted here. For the most part, the horrific tests were carried out on Chinese people, though prisoners-of-war, including men from Korea and Mongolia, were used.

For more than five years, General Ishii oversaw a wide range of experiments, many of them of dubious medical value to say the least. Thousands were subjected to vivisections, usually without anaesthetic. Often, these were fatal. Countless types of surgery, including brain surgery and amputations, were also carried out without anaesthetic. At other times, inmates were injected directly with diseases such as syphilis and gonorrhoea, or with chemicals used in bombs. Other twisted experiments included tying men up naked outside and observing the effects of frostbite, or simply starving people and seeing how long they took to die.

Once it was clear Japan was going to lose the war, General Ishii tried to destroy all evidence of the tests. He burned down the facilities and swore his men to silence. He needn’t have worried. Senior researchers from Unit 731 were granted immunity by the U.S. In exchange, they contributed their knowledge to America’s own biological and chemical weapons programs. For decades, any stories of atrocities were dismissed as ‘Communist Propaganda’. In more recent years, the Japanese government has acknowledged the Unit’s existence as well as its work, though it maintains most official records have been lost to history.

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“The Little Albert Experiment”

After many months observing young children, John Hopkins University psychologist Dr. John B. Watson concluded that infants could be conditioned to be scared of non-threatening objects or stimuli. All he needed was first-hand proof. Since it was 1919 and experimental ethics were nowhere near as strict as they are today, Watson, along with his graduate student Rosalie Rayner, set about designing an experiment to test their theory. Thanks to their connections at the Baltimore hospital, they were able to find a young baby, named ‘Albert’, and ‘borrow’ him for the afternoon. While Albert’s mother might have consented to her son helping out scientific research, she had no idea what Watson was actually planning.

The young Albert was just nine months old when he was taken from a hospital and put to work as Watson’s guinea pig. At first, Watson carried out a series of baseline tests, to see that the child was indeed emotionally stable and at the accepted stage of development. But then the tests got creepier. Albert was shown several furry animals. These included a dog, a white rat and a rabbit. Watson would show these toys to Albert while at the same time banging a hammer against a metal bar. This was repeated a number of times. Before long, Albert was associating the sight of the furry animals with the fear provoked by the loud, unpleasant noise. Indeed, within just a short space of time, just seeing the furry rat could distress the child.

Watson noted at the time: “The instant the rat was shown, the baby began to cry. Almost instantly he turned sharply to the left, fell over on [his] left side, raised himself on all fours and began to crawl away so rapidly that he was caught with difficulty before reaching the edge of the table.” The scientist and his research partner had achieved their goal: they had proof that, just as in animals, classical conditioning can be used to influence or even dictate emotional responses in humans. Watson published his findings the following year, in the prestigious Journal of Experimental Psychology .

Even at the time, Watson’s methods were seen as unethical. After all, isn’t a doctor supposed to ‘do no harm’? What’s more, Watson never worked with Little Albert again, so he wasn’t able to reverse the process. But still, the results were heralded as a breakthrough in our understanding of popular psychology. Notably, Watson recorded the Little Albert Experiment, and the videos can be seen online today. And, for what it’s worth, most experts now agree that, though he would have most likely feared furry objects for a short spell of time during his childhood, Little Albert probably lost the association between cute toys and loud noises.

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The “Monster” Study

These days, any tests carried out on children are subject to strict ethical rules and guidelines. This wasn’t the case back in the 1930s, however. So, when Wendell Johnson, a speech pathologist at the University of Iowa, wanted to carry out research on young participants, his institution was happy to oblige. Along with Mary Tudor, a grad student Johnson was supervising, work began in 1939. Over the next few years, dozens of kids would be subject to speech-related tests, with the effects of the experiment lasting for decades.

The purpose of the research sounded noble enough: Johnson wanted to see how a child’s upbringing affects their speech. In particular, he was fascinated by stuttering and determined to see what made one child stutter, yet another speak fluently. Thankfully, a local orphanage was able to ‘supply’ Johnson and Tudor with 22 children for them to work with. All of the young participants spoke without a stutter when they arrived at the University of Iowa labs for the first time. They were then divided into two equal groups, and the experiment got underway.

Both groups were asked to speak for the researchers. How they were treated, however, was completely different. In the first group, all of the children received positive feedback. They were praised for their fluent speech and command of the English language. The second group received the opposite kind of treatment. They were ridiculed for their inability to speak like adults. Johnson and Tudor would listen carefully for any little mistakes, and above all for any signs of stuttering, and criticize the children harshly for them.

Johnson’s methods shocked his academic peers. Not that they would have been so surprised. As a young researcher at the University of Iowa, he gained a reputation for experimenting with shock tactics. For instance, as a postgraduate student himself, Johnson would work with his colleagues trying to cure his own stutter, even electrocuting himself to see if that made a difference. But still, inflicting deliberate cruelty on children was seen as a step too far. As such, the Iowan academics nicknamed Johnson’s 1939 research ‘The Monster Study’. And the name was just about the only thing of significance it gave us.

With the University of Iowa keen to distances itself from news of human experimentation being carried out by the Nazis in war-torn Europe, they hushed-up the Monster Study. None of the findings were ever published in any academic journal of note. Only Johnson’s own thesis remains. The effects were clear, however. Many of the children in the second group went on to develop serious stutters. Some even had serious speech problems for the rest of their lives. The university finally acknowledged the experiment in 2001, apologising to those involved. Then, in 2007, six of the original orphan kids were awarded almost $1 million in compensation for the psychological impact Johnson’s work had on them.

Interestingly, however, while the methods used for the Monster Study have widely been condemned as being cruel and simply indefensible, some have argued that Johnson may have been onto something. Certainly, Mary Tudor said before her death that she and her research partner might have made serious contributions to our understanding of speech and speech pathology had they been allowed to publish their work. Instead, the experiment is now shorthand for bad science and a complete lack of ethics.

The Stanford Prison Experiment

Off all the ill-advised – and indeed, cruel – experiments North American universities have carried out over the decades, none is more infamous than the Stanford Prison Experiment. It’s so famous, in fact, that movies have been made based on the experiment which took place at Stanford University for one week in August 1971. Furthermore, while undoubtedly cruel, its findings are still used to inform popular understanding of psychological manipulation. Moreover, the behaviour of the participants involved is often held up as a warning about what can happen if humans are given power without accountability.

The experiment was led by Professor Phillip Zimbrano. As a psychologist, he was eager to see whether abuse in prisons can be explained by the inherent psychological traits of both guards and prisoners. Given the topic, he received funding from the U.S. Office of Naval Research. Funding in hand, Zimbrano set about recruiting participants. This turned out to be no problem at all, as a number of Stanford students volunteered to take part. Zimbrano then appointed some of the volunteers as guards and the others were designated as prisoners. The experiment could begin.

In the basement of the university’s psychology department, Zimbrano had built a makeshift ‘prison’. In all, 12 prisoners were kept here in small cells, while 12 guards were assigned a different part of the basement. While the prisoners had to endure tough conditions, the guards enjoyed comfortable, furnished quarters. The participants were also dressed for their parts, with the guards given uniforms and wooden batons. They were also kitted out with dark sunglasses so they could avoid eye contact with the people they were tasked with guarding.

Within 24 hours, any semblance of calm had gone. The prisoners started to revolt and the guards started to react. Special cells were set up to give well-behaving prisoners preferential treatment. The guards – who were barred from actually physically hitting their charges – started to use psychological methods to keep prisoners down. They would deny them food or put prisoners in darkened cells. Sleep was also denied to the prisoners. Within six days, Zimbrano agreed to halt the experiment. He did, at least, have more than enough evidence – some of it filmed – to draw on when making his conclusions.

Professor Zimbrano noted that around one third of the guards – again, young men taken randomly from the Stanford student population – exhibited genuine sadistic tendencies. At the same time, most of the inmates were seen to ‘internalise’ their roles. They took on the mentality of prisoners. While they could have left at any time, they instead gave up and became weak and passive. In the end, the experiment received, and continues to receive, criticism for the harsh methods used. Nevertheless, the findings of the Stanford Prison Experiment actually changed the way U.S. prisons are run and they are often held up as proof that most people can inflict cruelty and suffering on another human being if they are given a position of power and ordered to do so.

The South African ‘Aversion Project’

In Apartheid-era South Africa, national service was compulsory for all white males. At the same time, homosexuality was classed as a crime. Inevitably, therefore, any gay men who found themselves called into service were in for a tough time. But it wasn’t just name-calling or casual discrimination they had to contend with. Many were subjected to cruel experiments. The so-called ‘Aversion Project’, run throughout the 1970s and then the 1980s, was aimed at ‘treating’ homosexuals. As well as psychological treatments, it also used physical ‘treatments’, many of which would rightly be regarded as torture.

The project first really got started in 1969, with the creation of Ward 22. The creepily-named ward was part of a larger military hospital just outside of Pretoria and was designed to treat mentally-ill soldiers. For the unit chief Dr Aubrey Levin, this including homosexuals, regarded as unstable, or even ‘deviants’. For the most part, the doctor was determined to prove that electric shock therapy and conditioning could ‘cure’ the patients of their desires. Hundreds of men were electrocuted, often while being forced to look at pictures of gay men. The electric current would then be turned off and pictures of naked women shown instead in the hope that this would alter the mindset.

Inmates subjected to such experimental treatment would sometimes be tested, given temptations to see if they really were ‘cured’. Persistent ‘offenders’ were given hormone treatments, almost always against their will, and many were even chemically castrated. Even by the middle of the 1970s, when numerous, more ethical, studies had proven that ‘conversion therapies’ could change a person’s sexuality, Ward 22 carried on with its work. In fact, in only ended with the fall of the apartheid regime. To the very end of the project, Dr Levin maintained that all the men he treated were volunteers and asked for his help. Many of his peers disagreed, as did a judge, who sentenced him to five years in prison in 2014.

Project 4.1

On March 1, 1954, the United States carried out Castle Bravo , testing a nuclear bomb on the Bikini Atoll, in the middle of the Pacific Ocean. The test not only went without a hitch, it actually went better than expected. The yield produced by the bomb was much higher than scientists had anticipated. At the same time, the weather conditions in this part of the Pacific turned out to be different to what had been predicted. Radiation fallout from the blast was blown upwind, towards the Marshall Islands. But, instead of alerting the islanders to the danger, the project heads sensed an opportunity. How many times would they be able to see the affect of radiation fallout on a population for real?

Making the most of the opportunity, the American scientists simply sat back an observed. That is, they watched innocent people be affected by the fallout of an American nuclear bomb. Over the next decade, the project observers noted an upturn in the number of women on the Marshall Islands suffering miscarriages or stillbirths. But then, after ten years or so, this spike ended. Things seemingly returned to normal, and so scientists were unable – or unwilling – to make any formal conclusions. But then, things started to go downhill again.

At first, children on the Marshall Islands were observed to be growing less than would be expected. But then, it became clear that not only were they suffering from stunted growth, but a higher-than-expected proportion of youngsters were developing thyroid cancer. What’s more, by 1974, the data was showing that one in three islanders had developed at least one tumor. Later analysis, published in 2010, estimated that around half of all cancer cases recorded on the Marshall Islands could be attributed to the 1954 nuclear test, even if people never displayed any obvious signs of radiation poisoning in the immediate aftermath of the explosion.

Given that the initial findings of Project 4.1 as it was known were published in professional medical journals as early as 1955, the American government has never really denied that the experiment took place. Rather, what has been, and continues to be contested, is whether the U.S. actually knew that the islands would be affected before they carried out the test. Many on the Marshall Islands believe that Project 4.1 was premeditated, while the American authorities maintain that it was improvised in the wake of the explosion. The debate continues to rage.

The Tuskegee Experiments

For four decades, African-American men in Macon County, Alabama, were told by medical researchers that they had ‘bad blood’. The scientists knew that this was a term used by sharecroppers in this part of the country to refer to a wide range of ailments. They knew, therefore, that they wouldn’t question the prognosis. And neither would they raise any concerns or questions when the same researchers gave them injections. Which is how doctors working on behalf of the U.S. Public Health Service (PHS) were able to look on as hundreds of men went mad, blind or even died as a result of untreated syphilis.

When the experiment began back in 1932, there was no known cure for syphilis. As such, PHS researchers were determined to make a breakthrough. They went to Tuskegee College in Alabama and enlisted their help. Together, they enlisted 622 African-American men, almost all of them very poor. Of these men, 431 had already contracted syphilis prior to 1932, with the remaining 169 free from the disease. The men were told that the experiment would last for just six years, during which time they would be provided with free meals and medical care as doctors observed the development of the disease.

In 1947, penicillin became the recommended treatment for syphilis. Surely the doctors would give this to the men participating in the Tuskegee Experiment? Not so. Even though they knew the men could be cured, the PHR workers only gave them placebos, including aspirin and even combinations of minerals. With their condition untreated, the men slowly succumbed to syphilis. Some went blind, others went insane, and some died within a few years. What’s more, in the years after 1947, 19 syphilitic children were born to men enrolled in the study.

It was only in the mid-1960s that concerns started to be raised about the morality of the experiment. San Francisco-based PHS researcher Peter Buxton learned about what was happening in Alabama and raised his concerns. However, his superiors were unresponsive. As a result, Buxton leaked the story to a journalist friend. The story broke in 1972. Unsurprisingly, the public were outraged. The experiment was halted immediately, and the Congress inquiries began soon after. The surviving participants, as well as the children of those men who had died, were awarded $10 million in an out-of-court settlement. Finally, in 1993, President Bill Clinton offered a formal and official apology on behalf of the U.S. government to everyone affected by the experiment.

Project MK-Ultra

Though they had the Bomb, in the 1950s, the CIA were still determined to enjoy every advantage over their enemies. To achieve this, they were willing to think outside of the box. Perhaps the best example of this was MK-Ultra, a top-secret project where the CIA attempted to alter brain function and explore the possibility of mind control. While much of the written evidence, including files and witness testimonies, were destroyed soon after the experiments were brought to an end, we do know that the project involved a lot of drugs, some sex and countless instances of rule bending and breaking.

Project MK-Ultra was kick-started by the Office of Scientific Experiments at the start of the 1950s. Central to the project was determining how LSD affects the mind – and, more importantly, whether this could be turned to America’s advantage. In order to learn more, hundreds, perhaps even thousands of individuals, were given doses of the drug. In almost all cases, they were given LSD without their explicit knowledge or consent. For example, during Operation Midnight Climax in the early 1960s, the CIA opened up brothels. Here, the male clients were dosed up with LSD and then observed by scientists through one-way mirrors.

The experiments also included subjecting American citizens to sleep deprivation and hypnosis. Not all of the tests went plainly. Several people died as a direct result of Project MK-Ultra, including a US Army biochemist by the name of Frank Olsen. In 1953, the scientist was given a dose of LSD without his knowledge and, just a week later, died after jumping out of a window. While the official reason of his death was recorded as suicide, Olsen’s family have always maintained that he was effectively killed by the CIA.

When President Gerald Ford launched a special Commission on CIA activities in the United States, the work of Project MK-Ultra came to light. Two years previously, however, the-then Director of the CIA, Richard Helms, had ordered all files relating to the experiments to be destroyed. Witness testaments show that around 80 institutions were involved in the experiments, with thousands of people given hallucinogenic drugs, usually by CIA officers with no medical background. And so, in the end, was it all worth it? The CIA has acknowledged that the experiments produced nothing of real, scientific value. Project MK-Ultra has, however, lived on in the popular imagination and has inspired numerous books, video games and movies.

Guatemalan Syphilis Experiment

For more than two years in the middle of the 20 th century, the United States worked directly with the health ministries of Guatemala to infect thousands of people with a range of sexually transmitted diseases, above all syphilis. Since they wanted to do this without the study subjects knowing about it – after all, who would give their consent to being injected with syphilis? – it was decided that the experiment should take place in Guatemala, with soldiers and the most vulnerable members of society to serve as the guinea pigs.

The Guatemalan Syphilis Experiment (it was not given an official codename or even a formal project title) began in 1946. It was headed up by John Charles Cutler of the US Public Health Service (PHS). Despite being a physician himself, Cutler was happy to overlook the principle of ‘First, do no harm’ in order to carry out his work. Making use of local health clinics, he tasked his staff with infecting around 5,500 subjects. Most of them were soldiers or prisoners, though mental health patients and prostitutes were also used to see how syphilis and other diseases affect the body. Children living in orphanages were even used for the experiments.

In all cases, the subjects were told they were getting medication that was good for them. And, while all subjects were given antibiotics, an estimated 83 people died. In 1948, with the wider medical community hearing rumors of what was being done in Central America, and with the American government wary of the potential fallout, the experiments were brought to an abrupt end. Cutler would go on to carry out similar experiments in Alabama, though even here he stopped short of actually infecting his subjects with life-threatening diseases.

It was only in 2010, however, that the United States government issued a formal apology to Guatemala for the experiments it carried out in the 1940s. What’s more, President Barack Obama called the project “a crime against humanity”. That didn’t mean that the victims could get compensation, however. In 2011, several cases were put forward but then rejected, with the presiding judge noting that the U.S. government could not be held liable for actions carried out in its name outside of the country. A $1 billion lawsuit against the John Hopkins University and against the Rockefeller Foundation is still open.

Mengele’s Twins

A world at war gave the Nazi regime the ideal cover under which they would carry out some of the most horrific human experiments imaginable. At Auschwitz concentration camp, Dr Josef Mengele made full use of the tens of thousands of prisoners available to him. He would carry out unnecessarily cruel and unusual experiments, often with little or no scientific merit. And, above all, he was fascinated with twins. Or, more precisely, with identical twins. These would be the subjects of his most gruesome experiments.

Mengele would personally select prospective subjects from the ramps leading off the transport trains at the entrance to the concentration camp. Initially, his chosen twins were provided with relatively comfortable accommodation, as well as more generous rations than the rest of the inmate population. However, this was just a temporary respite. Mengele’s experiments were as varied as they were horrific. He would amputate one twin’s limbs and then compare the growth of both over the following days. Or he would infect one twin with a disease like typhoid. When they died, he would kill the healthy twin, too, and then compare their bodies.

Gruesomely, the records show that on one particularly bloody night, Mengele injected chloroform directly into the heart of 14 sets of twins. All died almost immediately. Another infamous tale tells of Mengele trying to create his own conjoined twins: he simply stitched two young Romani children back-to-back. They both died of gangrene after several long and painful days. Mengele also had a team of assistants working for him, and they were no less cruel.

Nobody will ever know just how many children or adults were victims of Mengele’s experiments. Despite being meticulous record keepers, the Nazis kept some things secret. Tragically for his victims and their relatives, Mengele never faced justice for his actions. He was smuggled out of Europe by Nazi sympathisers at the end of the war and lived for another 30 years, in hiding, in South America.

Where did we find this stuff? Here are our sources:

“Unmasking Horror: A special report.; Japan Confronting Gruesome War Atrocity”. Nicholas D. Kristof, The New York Times, 1995.

“Little Albert regains his identity”. American Psychology Association, 2010.

“Unit 731: Japan discloses details of notorious chemical warfare division”. Justin McCurry, The Guardian, April 2018.

“The Stuttering Doctor’s ‘Monster Study'”. Gretchen Reynolds, The New York Times, March 2003.

“The Real Lesson of the Stanford Prison Experiment” . Maria Konnikova, The New Yorker, June 2015.

“Gays tell of mutilation by apartheid army” . Chris McGreal, The Guardian, July 2000.

“Nuclear Savage: The Islands of Secret Project 4.1” . The Environment & Society Portal.

“Tuskegee Experiment: The Infamous Syphilis Study” . Elizabeth Nix, History.com, May 2017.

“The secret LSD-fuelled CIA experiment that inspired Stranger Things” . Richard Vine, The Guardian, August 2016.

“Guatemala victims of US syphilis study still haunted by the ‘devil’s experiment'” . Rory Carroll, The Guardian, June 2011.

“Nazi Medical Experiments” . The United States Holocaust Memorial Museum.

Articles on On-human experimentation

Displaying all articles.

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Ethics in human research

Jharna mandal.

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry - 605 006, India. E-mail: ni.oc.oohay@ajiraphsahbus

Srinivas Acharya

Subhash chandra parija.

‘Ethical conduct’ literally means simply doing the right thing, but in reality it means more. It involves acting in the right spirit, out of an abiding respect and concern for one's fellow creatures.

Human research is research conducted with or about people, or their data or tissues, with the sole intention to do good.

Human research involves significant risks and it is possible for things to go wrong. Despite the best of intentions and care in planning and practice, sometimes things go awry. Now and then mishaps may arise because of technical errors or an ethical insensitivity, neglect or disregard.

On rare occasions, the practice of research has even involved deliberate and appalling violation of human beings. Earlier, in the 1900s, there were no regulations regarding the ethical use of human subjects in research. There were no guidelines or any code drawn out for conduct and no Institutional Review Board (IRB). Here is a brief account of why rules and regulations were established and the need for all established research institutes to have an IRB became a necessity.[ 1 ]

THE NUREMBERG CODE

A well-known chapter in the history of research with human subjects opened on December 9, 1946, when an American military tribunal opened criminal proceedings against 23 leading German physicians and administrators for their willing participation in war crimes and crimes against humanity. Among the charges were that German physicians conducted medical experiments on thousands of concentration camp prisoners without their consent. Most of the subjects of these experiments died or were permanently crippled as a result.

As a direct result of the trial, the Nuremberg Code was established in 1948, stating that ‘The voluntary consent of the human subject is absolutely essential,’ making it clear that subjects should give consent and that the benefits of the research must outweigh the risks.

Although it did not carry the force of law, the Nuremberg Code was the first international document, which advocated voluntary participation and informed consent.[ 2 ]

THE DECLARATION OF HELSINKI

In 1964, the World Medical Association established recommendations guiding medical doctors in biomedical research involving human subjects. The Declaration governs international research ethics and defines rules for ‘research combined with clinical care’ and ‘non-therapeutic research.’ The Declaration of Helsinki was revised in 1975, 1983, 1989, and 1996, and is the basis for Good Clinical Practices used today.

Issues addressed in the declaration of Helsinki include:

• Research with humans should be based on the results from laboratory and animal experimentation
• Research protocols should be reviewed by an independent committee prior to initiation
• Informed consent from research participants is necessary
• Research should be conducted by medically / scientifically qualified individuals
• Risks should not exceed benefits

THE TUSKEGEE SYPHILIS STUDY (1932 – 1972)

One of the turning points in the development of a consensus for guidelines for ethical conduct in research was a project conducted by the US Public Health Service. Six hundred low-income, African-American males, 400 of whom were infected with syphilis, were monitored for 40 years. Free medical examinations were conducted; however, the subjects were not told about their disease. Even though a proven cure (penicillin) became available in the 1950s, the study continued until 1972, with participants being denied treatment. In some cases, when the subjects were diagnosed as having syphilis by other physicians, researchers intervened to prevent treatment. The study sparked off a wide-scale public outrage when it became publicly known, and the US government had to close it in 1973.

Due to the publicity from the Tuskegee Syphilis Study, a National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research was formed in the US, which was in charge of identifying the basic ethical principles that should underline the conduct of biomedical and behavioral research involving human subjects and to develop guidelines that should be followed, to assure that such research is conducted in accordance with those principles. The Commission drafted the Belmont Report, a foundational document for the ethics of human subjects’ research in the United States.[ 3 ]

THE BELMONT REPORT

The Belmont Report was published in 1979, with attempts to summarize the basic ethical principles identified by the Commission in the course of its deliberations. The Report is a statement of the basic ethical principles and guidelines that should assist in resolving the ethical problems that surround the conduct of research with human subjects. The three basic ethical principles and their corresponding applications according to the report are:

The Belmont Report established three basic ethical principles – respect for persons, beneficence, and justice – which are the cornerstones for the regulations involving human subjects.[ 3 ]

COMMON RULE

This is a set of regulations that have been adopted by many research agencies in the United States and elsewhere.

The main elements of the Common Rule include:[ 3 ]

• requirements for assuring compliance by research institutions
• requirements for researchers obtaining and documenting informed consent
• requirements for Institutional Review Board (IRB) membership, function, operations, review of research, and record keeping
• additional protection for certain vulnerable research subjects – pregnant women, prisoners, and children

Five Examples of Human Experimentation Leading to Scientific Breakthroughs

From dipping food into a bullet wound to swallowing a bacterial sample, these five examples of "human experimentation" opened new doors for science. Usually a scientist would never begin an experiment by testing in humans. After all, it would seem almost alien to start out immediately poking and prodding people based only on an educated hypothesis. Instead, researchers begin slowly in petri dishes, models or another simulated environment and move up from there.

However, there are times when a scientist has no other option. Believing in his or her own work so much, the only solution is to offer themselves up as a test subject to develop or prove something absolutely groundbreaking. It’s not taking the classical approach, but it gets the job done.

Five researchers make our list of scientists who conducted groundbreaking human experiments (or at least pushed ethical limits with humans) by experimenting on themselves, the unsuspecting or their willing partner.

1. William Beaumont :

Because of William Beaumont’s experimentation on Alexis St. Martin, he is coined "The father of gastric physiology;" however, some scientific historians question the ethics behind the research.

On June 6, 1822, Alexis St. Martin was accidentally shot in the torso. As an Army surgeon, Beaumont treated the wound, but expected St. Martin to die.

Instead, St. Martin recovered, but developed a fistula (open tube leading to his stomach) from his injury which left him unable to return to work. Beaumont hired him as a contracted servant, but found another opportunity for keeping St. Martin around. Due to the way the injury healed, Beaumont was able to observe St. Martin’s digestive processes. But here’s where it gets a little gross – to really gain insight into digestion, Beaumont would tie some string around a piece of food and insert it into the hole, observing it every few hours to see how well it had been digested. He also extracted gastric acid and continued experimentation after St. Martin left. Beaumont’s research led to an understanding that stomach acid plays a significant role in digestion and that chewing was not the primary process.

2. George Otto Gey :

Ok, this example doesn’t really involve experimenting on a person directly, but it is a famous case that seemed to defy human ethics. Gey was the scientist behind the development of the HeLa cell line, which is the first immortalized human cell line used for research. The reason it’s a touchy subject is because Henrietta Lacks, a cancer patient, was the unsuspecting source of these cells, and her surviving family received no financial gain.

In 1951 Lacks’s treating physician sent a biopsy to Gey’s lab. The cells grew at an astounding rate and were later sold to other researchers. The HeLa cell line contributed to other discoveries such as developing vaccines for human papillomavirus.

As fate would have it, Gey was diagnosed with pancreatic cancer. And as a committed researcher, right before an emergency surgery, he instructed doctors to take a biopsy and attempt to grow another line of cells for research. Unfortunately, his wishes were ignored.

3. Science for the Masses (SFM):

In 2015, a group of independent researchers, described as “biohackers,” collaborated on a project to test the possibility of night vision. Using information from Totada R. Shantha’s 2012 patent for using Chlorin e6 (Ce6) as a treatment for night blindness, the Science for the Masses team set out to experiment on one of their own. Gabriel Licinia offered himself to be the test subject, while Jeffrey Tibbetts, another SFM member, pipetted 50 microliters of their Ce6 mixture into the conjunctival sack of Licinia’s eye. Their mixture was based on the original patented formula; however, the SFM team added insulin and dimethylsulfoxide to improve the blend. The experiment worked.

When tested at night in the woods, Licinia was able to identify people and shapes at given distances 100% of the time, while the control group was only able to identify objects one third of the time. This experiment, however, was meant for informational purposes. And the SFM team cautions others not to try this on their own since increasing light amplification might cause negative side effects. With that said, the SFM team also said it may open opportunities to grant soldiers improved night vision.

4. Jonas Salk:

While Jonas Salk followed conventional experimental methods, he makes the list since he and his family were among the initial test subjects. Salk is famed for developing a polio vaccine using a “killed-virus.” Originally, when proposing the idea, he was insulted by other researchers, even called “a mere kitchen chemist” by virologist Albert Sabin. Despite some of the negative press, Salk was developing his vaccine faster than developers of live-virus vaccines, and March of Dimes resources soon went to support his endeavor.

After effectively inoculating monkeys first, he volunteered himself and family for the next step. In 1952, Salk boiled some needles in his kitchen stove and administered the vaccine to himself, his wife and three kids. Around that same time, he also partnered with the D.T. Watson Home for Crippled Children and the Polk State School, administering the vaccine to a small sample of children. The results were successful, and after gaining more public support, one of the biggest American clinical trials began. Between April and of June 1954 there were 1.8 million “polio pioneers.”

But even more heroic than volunteering himself and family to literally save millions, Salk opted not to patent the vaccine and received no compensation for his discovery. Salk was once asked about who owned the patent, and he replied, “Well, the people, I would say. There is no patent. Could you patent the Sun?”

5. Barry J. Marshall and J. Robin Warren:

We started with the stomach, and we’re ending with the stomach with Barry Marshall and J. Robin Warren. This is perhaps the best example of self-experimentation leading to brilliant discoveries.

Warren was a pathologist who had been studying gastritis, which can lead to stomach ulcers and gastric cancer, and Marshall began taking an interest in the work. The two noticed that the usual drug treatments that blocked gastric acid would only work temporarily, and patients would often relapse. Together they began to study a spiral bacterium ( Heliobacter pylori ) that appeared to be associated with stomach ulcers. However, the suggestion that bacteria could live in the acidic conditions of the stomach seemed preposterous to most scientists. To make things more difficult for Warren and Marshall, during their research, the pair was unable to infect piglets to prove their theory. But being so sure of their research, Marshall drank from a dish containing cultured H. pylori . Of course he did not do this before having an endoscopy to show his gastric conditions were normal. After ingesting the culture, Marshall began experiencing the initial symptoms: nausea and halitosis, which crept up only three days later. Five days later he began vomiting. And on the eighth day, Marshall had a second endoscopy and biopsy, revealing he had gastritis and H. pylori was present. To counter the infection, Marshall began taking antibiotics.

Their risky move was rewarded in 2005 with a Nobel Prize in Physiology or Medicine for the discovery of Heliobacter pylori and its role in gastritis and peptic ulcer disease.

Do you think any other scientists make the list? Comment below and share their stories!

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Investigating the effects of microgravity, magnetic, and fungal influences on root growth of low-Earth orbit germinating plants to help inform plant growth procedures for food viability and sustainability in lunar or Martian gravitational environments as well as deep-space exploration. Additionally, the research will provide actionable physical and genetic data relative to plant growth utilizing gravitropism, fungal volatiles, and magnetic fields to improve food production here on Earth.

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• Published: 27 June 2024

First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

• Stefana Orobeti 1 , 2   na1 ,
• Livia Elena Sima 2   na1 ,
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• Constantin Diplasu 1 ,
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• Gabriel Cojocaru 1 ,
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Scientific Reports volume  14 , Article number:  14866 ( 2024 ) Cite this article

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• Laser-produced plasmas
• Radiotherapy
• Wide-field fluorescence microscopy

Radiation delivery at ultrahigh dose rates (UHDRs) has potential for use as a new anticancer therapeutic strategy. The FLASH effect induced by UHDR irradiation has been shown to maintain antitumour efficacy with a reduction in normal tissue toxicity; however, the FLASH effect has been difficult to demonstrate in vitro. The objective to demonstrate the FLASH effect in vitro is challenging, aiming to reveal a differential response between cancer and normal cells to further identify cell molecular mechanisms. New high-intensity petawatt laser-driven accelerators can deliver very high-energy electrons (VHEEs) at dose rates as high as 10 13  Gy/s in very short pulses (10 –13  s). Here, we present the first in vitro experiments carried out on cancer cells and normal non-transformed cells concurrently exposed to laser-plasma accelerated (LPA) electrons. Specifically, melanoma cancer cells and normal melanocyte co-cultures grown on chamber slides were simultaneously irradiated with LPA electrons. A non-uniform dose distribution on the cell cultures was revealed by Gafchromic films placed behind the chamber slide supporting the cells. In parallel experiments, cell co-cultures were exposed to pulsed X-ray irradiation, which served as positive controls for radiation-induced nuclear DNA double-strand breaks. By measuring the impact on discrete areas of the cell monolayers, the greatest proportion of the damaged DNA-containing nuclei was attained by the LPA electrons at a cumulative dose one order of magnitude lower than the dose obtained by pulsed X-ray irradiation. Interestingly, in certain discrete areas, we observed that LPA electron exposure had a different effect on the DNA damage in healthy normal human epidermal melanocyte (NHEM) cells than in A375 melanoma cells; here, the normal cells were less affected by the LPA exposure than cancer cells. This result is the first in vitro demonstration of a differential response of tumour and normal cells exposed to FLASH irradiation and may contribute to the development of new cell culture strategies to explore fundamental understanding of FLASH-induced cell effect.

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Introduction.

New radiotherapeutic treatment schemes for solid cancers have been developed and tested to provide a differential response of targeted tumour cells compared with that of normal cells. Radiotherapy delivered via ultrahigh dose rate (UHDR) irradiation, known as FLASH, has demonstrated similar efficacy in affecting tumour progression to conventional dose rate radiotherapy while reducing the toxicity of the healthy tissue encountered in standard treatment 1 , 2 . FLASH irradiation is defined by dose rates greater than 40 Gy/s; this is a few orders of magnitude greater than those of conventional treatments carried out at doses of approximately 0.03 Gy/s. The FLASH-induced effect was validated in preclinical experiments with electron 3 and proton beams 4 . However, the biological mechanisms underlying the FLASH effect are poorly understood. FLASH irradiation may cause the consumption of oxygen around the tumour and produce temporary local hypoxia, reducing the development of fast-proliferating cancer cells 5 . Other studies proposed that FLASH irradiation may be involved in the reduction of hydrogen peroxidase, which is correlated with the modification of reactive oxygen species (ROS) production in tissues and affects the biological response 6 . In the last decade, several laboratories have modified linear accelerators to generate UHDRs and studied the FLASH effect 1 , 7 . To date, the UHDR-induced FLASH effect has been demonstrated in several in vivo cancer animal models.

Compared with photon-based therapy, the use of very high-energy electrons (VHEEs) with energies greater than 50 MeV is an alternative therapeutic solution for deep-seated tumours due to the potential improvement in the dose distribution. This potential improvement has been intensively explored in recent years and new, more effective radiotherapeutic strategies were developed 8 , 9 , 10 .

High-intensity laser-driven acceleration of VHEE represents a new approach that may be an alternative solution for radiotherapeutic treatments with electrons. Indeed, VHEEs with dose rates as high as 10 13  Gy/s 11 , 12 , 13 , 14 are achievable by laser-plasma accelerators (LPAs) with quasi-monoenergetic beams 15 . This ultrafast radiation biology may be considered a new emerging interdisciplinary field, in combination with advances in ultrashort radiation sources, high-energy chemistry, and anticancer therapy 16 . The unique characteristics of LPA sources rely on its wider accessibility with respect to conventional accelerators, and LPA sources have practical advantages that could have a major impact on the development of new radiotherapeutic sources. Notably, an LPA system capable of producing electrons with energies of hundreds of MeV exhibits a footprint of tens of square metres, which is much less than that of a similar conventional linear accelerator (LINAC). In addition, a few hundred centimetres of an electron pathway demonstrates the system's ability to provide a table-top compact setup, which is very useful for radioprotection procedures. These electron beams were found to be suitable for delivering a high dose peak on the propagation axis with deep penetration combined with a sharp and narrow transverse penumbra 17 . Initial studies with LPA were devoted to characterizing dosimetric aspects 18 , 19 , 20 , 21 , 22 .

LPA’s has been proposed as a new platform for VHEE-induced FLASH irradiation 15 , but very few experiments have explored the feasibility of this technique 23 . The first irradiation experiments for in vivo studies involved more than 50 mice, using a dose rate exceeding 1 Gy/min at electron energies above 5 MeV; this energy is considered stable and provides reliable operation of the system 24 . In addition, only a few other studies have attempted to assess the effects of LPA VHEE on cell damage. LPA VHEE was shown to cause DNA damage in skin carcinoma cells exposed to a very high dose rate from a single, ultrashort bunch of high-energy electrons 16 . Additional experiments with such short durations of LPA beams may help discover novel phenomena in radiobiology, particularly at the early phase of the damage induced to cell structure by ionizing radiation.

Here, we present the first in vitro experiments carried out with cell co-cultures exposed to LPA electrons generated by the interaction of a high-intensity laser beam with gas jets. Melanoma cancer cells and normal melanocyte co-cultures were irradiated with either VHEE produced by LPA or pulsed X-ray beams. The potential differential responses of cancer cells and normal cells to the proposed irradiation setup available in our facility were evaluated by measuring induced double-strand DNA breaks in comparison to X-rays. First step necessary to determine if a certain radiation source and applied protocol has the potential to become one of the elements in the oncologists’ toolbox to be used in the fight against tumours is to evaluate its DNA damage inducing capacity 25 . This step was crucial for demonstrating the quality potential of VHEEs generated by LPA, thus faciliting new opportunities for extensive utilization in clinical studies.

Here, we present the results obtained in vitro in two irradiation sessions in which the conditions were similar, and these sessions are further referred to as experiments PW1 and PW2. The electron spectrum was quasi-monoenergetic with a peak energy E peak of ~ 190 ± 40 MeV (Fig.  1 b). Values under 50 MeV are missing due to the constructive spectrometer limit. The number of applied laser shots varied from one experiment to another, and a fixed total dose of 150 mGy was maintained. This dose was measured using an ionization chamber located 5 cm behind the stack of containers and thermoluminescent dosimeters (TLDs) (as depicted in Fig.  4 ). The array of images presented in Fig.  1 a shows the LANEX images acquired during a session in which 21 shots were applied. The laser energy was highly stable over the 21 shots, with an E laser (mean ± s.d.) = 7.02 ± 0.34 J (see Fig.  1 c and movie in the Supplementary Information).

( a ) Raw images of the scintillating screen together with the cell containers exposed to laser-plasma accelerated electrons; ( b ) Energy spectrum of electrons; ( c ) Laser energy stability during the 21 shots session.

The doses measured by the three independent dosimetry systems for each of the two (I, II) irradiation sessions were as follows: (i) I. 149.9 mGy and II. 154.8 mGy by the ionization chamber; (ii) I. 1107.28 mGy, 627.48 mGy, and 400.97 mGy; II: 1263.08 mGy, 748.28 mGy, and 429.65 mGy by the three TLDs placed in front, middle, and behind the cell containers, respectively; and (iii) I. 195 mGy and II. 170 mGy by Gafchromic films (GFs) placed behind the stack. The differences in the measured dose were potentially the result of the broad energy spectrum of the electron beam accelerated by high-power laser target interactions. The highest values recorded by the TLD readings (highest low limit sensitivity) potentially indicate the presence of low-energy electrons transient through and/or stopped within the samples and not registered by the Gafchromic films and Advance Markus ionization chamber. A discussion on this aspect is presented in the SI (Supplementary Information). Additionally, the three dosimetry systems were placed at different depths along the electron beam propagation axis and involved different radiation-sensitive media, which accounts for the variations in dose.

We measured the immediate response of the radiation-resistant A375 melanoma cells and normal human epidermal melanocytes (NHEMs) 30 min after the exposure of cocultures to irradiation treatments, when p-γ-H2AX was shown to have its peak nuclear expression at DNA double-strand break (DNA-DSB) sites 26 . For this, two SlideFlasks containing NHEM: A375 co-cultures (seeded one day prior to the experiments in a 1:2 numeric ratio) were irradiated serially at an ultrahigh dose rate (UHDR) within the same session using the PW laser facility, while a third identical SlideFlask was irradiated using an optimized pulsed X-ray setup with a conventional dedicated device, and a forth remained non-irradiated. GFs were attached to the bottom of each slide to record the dose and pattern of exposure of each replicate. After 30 min of incubation at 37 °C, the cells were fixed for immunofluorescence analysis of the induced DNA damage foci. In parallel, a sample was treated for 24 h with 2 μM cisplatin (CisPt) to serve as a positive control for foci induction (Figure SI 3 , A–C). DNA DSBs are produced in response to stress-induced either by chemical ( e.g ., chemotherapeutics, such as CisPt) or radiological ( e.g., X-ray exposure) treatments. If not resolved by cell molecular repair mechanisms, the affected cells are stalled from proliferation and later die by the induction of apoptosis or alternative death pathways 27 . Hence, the occurrence of DNA damage foci is the first consequence of effective stress inflicted upon the targeted cells. We sampled 3 areas from the top, centre, and bottom of the vertically positioned slide to analyse the effect produced by PW laser-induced electron irradiation (Fig.  2 a and c; the developed GF vs . TissueFAXS scanned preview). Upon scanning the ROIs with a high magnification (63 ×) objective, the number of p-γ-H2AX foci in each nucleus was quantified using an image cytometry approach (Fig.  3 ). Notably, the DNA damage effect observed by foci quantification in each of the 3 selected regions of each PW irradiation exposed SlideFlask (Figs.  4 and 5 ) was consistent with the gradient pattern detected by GF dose mapping (Fig.  2 a and c; the developed GF vs. TissueFAXS scanned preview).

Analysis of the A375 melanoma and NHEM co-cultures upon laser-plasma accelerated electron exposure during 2 sequentially performed irradiation experiments (PW1, PW2) versus pulsed X-ray irradiation. ( a , c ) Correspondence between the irradiated GF fingerprint and selected areas of the SlideFlasks for image cytometry analysis (5 × magnification preview mode). ( b , d , e , f ) DNA damage response revealed as p-γ-H2AX foci at 30 min post-exposure to PW or X-ray-generated radiation versus non-exposed (untreated) or mock-exposed (PW env) negative controls. Immunofluorescence microscopy images of overlapping DAPI and TxRed signals (top), as well as grayscale images of the TxRed signal (bottom) of a representative field of view (FOV) under each irradiation condition, are shown. The p-γ-H2AX foci are visible in the detailed insets ( b , d , f —bottom right). Scale bar = 20 μm ( b , d , f ) or 100 μm ( e ).

Image cytometry analysis strategy of irradiated A375:NHEM co-cultures. Upon labelling with antibodies against p-γ-H2AX (red) and with Hoechst for nuclear DNA (blue), samples were automatically scanned using the TissueFAXSiPlus imaging system. After regions of interest (ROIs) reconstitution out of stitched individual fields of view (FOV), the obtained images were imported into the TissueQuest image quantification software. Each cell was identified based on nuclear DAPI signal by the segmentation algorithm. Next, scattergrams were generated based on DAPI-Eccentricity versus DAPI-Mean Intensity profiles and cell lines differentiated based on nuclear elongation (eccentricity): elongated nuclei of NHEM were gated (red gate) based on higher eccentricity values, while non-elongated A375 nuclei were gated (blue gate) based on low eccentricity values. Using forward gating, one can identify the scattergram displayed signals for each cell in a population. Here, we exemplify a NHEM nucleus encircled in red and an A375 nucleus encircled in green and their corresponding signals in the scattergrams (high vs. low elongation). In order to quantify p-γ-H2AX mean fluorescence intensity (MFI) and number of foci per nucleus, TxRed signals from the nuclear mask were quantified using 2 parameters: Texa-Mean Intensity and Texa_DOTS_ON_DAPI-Dots Count, respectively, visible on the axes. Histograms displaying frequency distribution of number of foci per nucleus are displayed in blue for each cell population. Also, scattergrams of number of foci versus p-γ-H2AX expression level can be generated. Here, red bars and dots identify the selected nuclei in the picture. Foci are indicated by white arrows (in NHEM) or arrowheads (in A375).

Frequency distribution of number of foci per nucleus for cells in each region of interest (ROI) analysed of the X-ray ( a ), PW1 ( b ), and PW2 (c) irradiated samples. Cells with 0 up to 44 foci per nucleus were identified in the irradiated and non-irradiated samples (Texa_DOTS_ON_DAPI – Dots count parameter). A threshold is depicted as baseline detected in normal melanocytes (NHEM) in the absence of radiation exposure (a—top left). One can observe an increase in the fraction of cells presenting higher number of p-γ-H2AX foci per nucleus in samples exposed to different irradiation conditions ( a – c ), as compared to non-irradiated control (a – top histograms) (n = 4473–10,629 analysed nuclei).

Comparison of “number of p-γ-H2AX foci per nucleus” values for each tested irradiation condition. The bar graphs show the comparisons between treatments for the overall A375 and NHEM co-cultures ( a ), for the A375 cells in co-cultures ( b ), and for the NHEM cells in co-cultures ( c ). Sampled areas of PW-irradiated specimens and the standard pulsed X-ray-irradiated cells were compared to non-irradiated controls. Medians of each data set and 95% confidence intervals are displayed. The statistical differences were determined using the nonparametric Mann–Whitney test, (n = 4473–10,629 analysed nuclei, * p  < 0.05, *** p  < 0.001, **** p  < 0.0001, ns  = not significant).

The first irradiation procedure (sample PW1) induced the greatest effects in the centre of the slide (Fig.  2 b, Fig.  4 b and Fig.  5 a), while the second (sample PW2) procedure impacted mostly the top of the irradiated sample (Fig.  2 d, Fig.  4 c and Fig.  5 a). Consequently, these areas had an increased frequency of nuclei with high foci counts compared to the other two regions in the corresponding samples (Fig.  4 and Fig.  5 a). The top area of the PW2 slide had a greater tendency for DNA-damaged nuclei than the other investigated areas; the centre of the PW1 slide showed the most comparable effect to that of X-ray irradiation (Fig.  2 b vs. f., Fig.  4 b vs. a, and Fig.  5 a, cell co-culture), and this was considered to be a positive control for radiation-induced DNA-DSB. Interestingly, upon analysing each cell type individually (Fig.  5 b and c, respectively), we observed that PW2 exposure had a different pattern of impact on NHEMs than on the A375 melanoma cells; here, the normal cells were affected more in the centre area of the slide than in the top area, when compared to cancer cells. This was observed only for this irradiation condition. Hence, NHEM cells had increased DNA damage in the centre of the slide, irrespective of the PW1 and PW2 irradiation patterns. The environment in the PW room did not induce supplementary DNA damage compared to the baseline observed in untreated control cells (Fig.  2 e).

Approximately 50% of all cancer patients undergo a radiotherapeutic regimen 28 . Radiotherapy is currently applied intensively in the management of solid tumours, alone or in combination with other chemotherapy or immunotherapy treatments. As a new radiotherapeutic strategy, the FLASH effect induced by UHDR irradiation has been demonstrated in several in vivo animal models 29 . In order to produce a fundamental understanding of FLASH irradiation and beam interactions with cells, in vitro models need to be developed and investigated. In addition, the in vitro setup may be more reliable for certain experiments than animal testing since human cells can be used and the tumour microenvironment can be replicated under controlled conditions. The demonstration of the FLASH effect in vitro is challenging, but necessary in order to reveal a differential response between cancer and normal cells and identify the underlying molecular mechanisms involved. Phosphorylated γ-H2AX is the most sensitive marker of DNA damage and repair 30 . DNA methylation can rapidly reveal DNA double-strand breaks (DNA-DSBs), which are considered the most lethal form of DNA damage that compromises genomic stability 31 . Therefore, p-γ-H2AX foci analysis is the most robust method for investigating DNA damage and repair, while foci quantification is known as an accurate radiation biodosimeter 32 .

For the conventional X-rays used in radiotherapy, the molecular mechanisms involved in the generation and repair of DNA-DSBs are fairly well understood. Upon irradiation, the most damaging lesion produced is DNA-DSB. Genes involved in the cell cycle and growth control are upregulated 33 , and the progression of the cell cycle is stalled to allow the initiation of the DNA repair mechanism. One of the initial events upon DNA damage consists of the phosphorylation of thousands of γ-H2AX molecules flanking the damaged sites; these can be microscopically detected as foci. The radiation dose-dependent γ-H2AX phosphorylation reaction peaks 30 min after exposure and then declines over the following hours during the process of DNA repair 26 . The subsequent repair process is supported by the activation of key signalling molecules and pathways regulating post-irradiation cell fate. In addition to its role in activating antiapoptotic signalling, Akt stimulates DNA-DSB repair in tumour cells through the nonhomologous end-joining (NHEJ) repair pathway, thereby promoting tumour cell survival post-irradiation 34 . If DNA damage is persistent and intolerable by the cell, one of the death pathways will become activated 27 . The persistence of substantial numbers of γ-H2AX foci for 48 h after application of irradiation doses greater than 1 Gy allows the precise estimation of patient exposure when analysing peripheric blood lymphocytes or sampled skin 32 .

However, less is known on the irradiation dose ranges, fractionation regimens, and underlying mechanisms that can induce DNA damage and tumour cell death in regard to novel irradiation technologies. There are various pathways of regulated cell death (reviewed in 35 ), and many were found to be involved in the tumour cell response to radiation 36 , such as senescence 37 , apoptosis 38 , necroptosis 39 and necrosis 40 , 41 . The mode of tumour cell death is crucial in vivo for determining whether anti-tumour immune priming occurs (known as the abscopal effect of radiotherapy) 42 . This systemic immune response triggered by local radiotherapy functions as an in situ cancer vaccination that contributes significantly to disease control.

Pioneering studies demonstrated that protonation accelerated by LPA resulted in the distinct formation of γ-H2AX foci at a proton dose of 20 Gy 43 and dose-dependent biological damage induced in vitro in tumour cells 44 . On the other hand, the understanding of the DNA damage process in cells after electron irradiation delivered by LPA is still very limited 45 , 46 . Exposure to 100 fs single-shot 1 Gy of electron at a mean energy of 95 MeV demonstrated DNA damage in irradiated carcinoma cells according to the comet tail test. A very high dose rate demonstrated that a measurable assessment of the immediate and reversible DNA damage in cells could be performed at a single-cell level 16 . In another pioneering study, cancer cells exposed to electron bunches generated by LPA with an exponential spectrum of an average energy of 1.5 MeV at a cumulative dose of up to 2 Gy showed a radiobiological response by the induction of micronuclei and shortening of telomeres, as well as by the reduction of cell survival in blood samples and cancer tissue 47 .

Our studies represent the first in vitro experiments with cell co-cultures exposed to LPA electrons generated by the interaction of a high-intensity laser beam with gas jets. The cells were analyzed by immunofluorescence microscopy to quantify the DNA damage foci in response to the stress induced by radiological treatments using LPA electrons compared to pulsed X-ray irradiation. A non-uniform dose distribution on some discrete areas was revealed by Gafchromic films (GFs) in the case of LPA electron beam exposure. We detected double-strand DNA breaks in response to LPA electron radiation exposure. Using microscopic p-γ-H2AX foci counts, we showed that the number of foci induced 30 min after irradiation was consistent with the level of exposure, as revealed by dose mapping (Fig.  2 ); an increased frequency of nuclei with a high number of foci was detected in the sampled co-culture areas most affected by irradiation (Fig.  4 ; Fig.  5 ; Figure SI 4 ). In addition, in several investigated areas, more DNA-damaged cell nuclei were generated by LPA electrons than by X-ray irradiation at a cumulative dose one order of magnitude lower. Additionally, the nuclei exposed to the highest doses of the LPA electrons exhibited an increased number of DNA damage foci, as opposed to those exposed to pulsed X-rays. Next, we sought to investigate if a differential response of normal cells versus cancer cells occurred when exposed to the radiation source; if present, this differential response could account for the FLASH effect.

The benefit of radiotherapy is presently limited by the tolerance limit imposed by the normal organ tissues to increased doses of applied radiation. For example, radiation therapy was excluded from the list of treatment options for patients with intra-abdominal tumours (such as ovarian cancer-OC), due to high toxicity inflicted on the intestine, upon exposure to total abdominal irradiation 48 . Currently, there is a revived interest in the potential use of abdominal FLASH irradiation for the treatment of OC. Studies in mice have shown that it provides similar efficacy to conventional RT in controlling peritoneal metastases, while preserving intestinal function 49 . Moreover, the same team showed that abdominopelvic FLASH RT improves immunotherapeutic efficacy of anti-PD-1 checkpoint inhibition in preclinical models of OC 50 . A recent study evaluated the impact of UHDR FLASH total body irradiation (TBI) on blood cancers and normal haematopoiesis in a humanized mouse model 51 . The authors investigated T-cell acute lymphoblastic leukaemia (T-ALL) progression and normal human haematopoiesis, upon FLASH-TBI versus CONV-TBI; FLASH (4 Gy) was produced using a prototype 6 MeV electron beam linear accelerator. Leukaemic cells extracted from irradiated mice were either cultured in vitro or transplanted into secondary recipients. The number of proliferating cancer cells was 4 times lower for FLASH-RT than for CONV-RT-exposed cells 7 days after culture; the leukaemic tumour burden was significantly decreased in secondary recipients, with 6% of leukaemic cells (CD45 + CD7 + ) in the bone marrow (BM) of mice bearing FLASH-RT cells and > 90% of leukaemic cells in mice receiving CONV-RT-exposed cells. Haematopoiesis and normal haematopoietic stem/progenitor cell functions, were partially spared upon FLASH-RT irradiation, while CONV-RT completely abolished these functions. Hence, the detrimental effects of CONV-RT on normal cells were reduced by using FLASH-RT setup, while increasing the benefit of radiation treatment 51 . Interestingly, when secondary transplantation was performed from a mixture of leukaemic and normal human haematopoietic cells, FLASH-RT was superior in preventing cancer relapse but impeded normal haematopoiesis from occurring, potentially due to alteration of the BM niche in the humanized mouse model 51 . Normal tissue sparing by FLASH-RT in the UHDR regimen has been previously demonstrated in another acute responding organ (gastrointestinal tract), as well as in late responding organs (lung, brain, skin), relatively independent of the ionizing radiation source type 52 , 53 .

In our present study, we concomitantly exposed melanoma cancer cells and normal melanocytes as in vitro co-cultures to FLASH irradiation via VHEE generated by the interaction of a high-intensity PW laser beam with a supersonic gas jet. The differential responses of cancer and normal cells were detected in discrete areas of the exposed specimen. Notably, we identified a discrete sample area where the VHEE exposure conditions had less DNA damage-inducing impact on the non-cancerous NHEM cells than on the A375 melanoma cells (Fig.  5 – top area of the PW2-irradiated slide). This result may be an essential step towards the possibility of understanding the fundamental mechanisms of the FLASH effect for our PW laser electron beam generation system. However, further VHEE setup optimization is needed to enhance the system stability and enable experimental reproducibility to determine an accurate correlation between the experimental parameters and cell responses. This phase is key for validating the potential of the VHEE generated by LPA and can be further developed and potentially tested in clinical trials.

Recent results obtained with VHEE produced by an LPA accelerator in water phantoms have shown that a significant dose relevant for preclinical studies can be delivered to deep-seated targets 23 . The cumulative dose delivered to the target was up to 1.6 Gy and was achieved by applying a few hundred shots. Thus, deep-seated tumours could be targeted by VHEE beams, as the dose deposited in the tissue can be higher than doses currently delivered by conventional radiotherapies 46 . Furthermore, the dose distributions generated by VHEE electrons are less affected by body inhomogeneities than those generated by protons 54 . Although FLASH-RT technology is still in its infancy from the viewpoint of medical application, the first case studies on human patients have been reported 55 , 56 , and three phase 1 clinical trials are ongoing to test primarily toxicity in patients with thoracic bone metastases or melanoma (reviewed in 57 ). However, the knowledge gap remains important, and safe and efficient parameters need to be determined for translation into the clinic and for the generation of a positive response. To address this issue, extensive research is needed to understand the dose-dependent mechanisms of action of electron FLASH-RT on cells 58 and to determine which are the prerequisite patient characteristics that classify patients as responders. The studies of Chabi S and collaborators 51 presented above revealed intrinsic genetic cues that differentiated radiation-sensitive from radiation-resistant leukaemic cancer cells.

In perspective, ultrafast radiation biology will likely be developed as a new domain in an emerging interdisciplinary field, along with the development of new ultrashort radiation sources and advanced cancer therapeutic strategies. Moreover, very high dose rates and ultrashort dose fractionations may be proposed in combination with advanced chemotherapeutic strategies because they enable real-time control of the amplified radio-sensitivity 59 .

Our results represent the first demonstration of a differential response of tumour and normal cells exposed the FLASH irradiation using an in vitro model. The experiments confirm the DNA damage-producing capacity of the laser-driven high-energy–dose electrons produced by a table top high power laser – plasma accelerator in cancer cells for future FLASH-RT applications. These findings may contribute to the development of either new cell culture approaches to explore fundamental understanding of cell induced FLASH effect or to the employment of new strategies for the improvement of stability and uniformity of new FLASH irradiation sources.

Materials and methods

Cell culture.

Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively. The A375 cells were grown in high-glucose (HG) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-Glutamax, and 1% penicillin/streptomycin; all reagents were purchased from Thermo. The NHEM cells were grown in MBM-4 medium (#CC-3250) supplemented with SingleQuots (#CC-4435) and manipulated according to the manufacturer’s instructions. The cells were maintained in a humidified incubator with 5% CO 2 at 37 °C. One day before the irradiation experiments, the cells were plated in 9 cm 2 polystyrene SlideFlasks (#170920, Thermo) in a 1:2 ratio of NHEM:A375 cells (420,000:840,000 cells) and the corresponding 1:1 media mix; the cells reached confluency within 24 h. Immediately before irradiation, the SlideFlasks were filled with 19 mL of prewarmed complete DMEM HG media. For cell exposure, we used SlideFlask containers, as they provide a high number of cells on large areas for exposure to VHEE or X-ray irradiation and the possibility of removing the flask chamber from the slide supporting the cells to be analysed with high resolution by fluorescence microscopy. For PW room environmental exposure condition testing, the cells were seeded directly onto the plastic surface of 24-well plates (Corning) and placed in the irradiation room outside the beam area during the experiment (PW env), and another well plate was left in the incubator (untreated). The technical control samples were seeded on 12 mm coverslips (Marienfeld) plates and placed in 24-well plates (Corning); the untreated and 2 μM cisplatin (CisPt)-treated cells were compared to set the thresholds for the analysis of the DNA damage-induced foci.

The high-intensity laser beam was focused with an off-axis parabolic mirror with a 3.2 m focal length (f# 27) on a supersonic gas jet consisting of 99% He + 1% N 2 60 . The quasi-monoenergetic electron energy distributions were measured with a spectrometer equipped with a Pb collimator and magnetic dipole of 0.8 T, which was set up into the electron beam at the extension of the interaction chamber. Figure  6 shows a sketch and an image of the experimental setup used for LPA VHEE cell irradiation (LPA experimental setup and photo in Figure SI 1). Three irradiation sessions were performed. Two experimental configurations were chosen to allow simultaneous irradiation of the two containers with cells grown either in the first or both containers; Fig.  6 shows the configuration in which cells were placed only in the first container. In our set-up, three thermoluminescent dosimeters (TLDs) were introduced: one was placed in front of the first container, the second was placed between the two containers, and the third was placed after the second container (see TLDs 1, 2, and 3 in Fig.  6 ). Before reaching the first TLD, the electron beam travelled approximately 198 cm in vacuum, and then it travelled successively through a 60 µm Al foil, a 1 cm thick glass window, another 60 µm Al foil, 0.5 mm cardboard, the scintillator screen LANEX and 2 cm in air.

Laser-gas target experimental setup for the generation of the accelerated electron beams and irradiation of the cell co-cultures in vitro.

Dosimetry measurements

Electron energy distribution measurements were carried out using a spectrometer equipped with a collimator and magnetic dipole, and the sample was placed into the electron beam at the extension of the interaction chamber. To estimate the dose, we used three independent dosimetry systems: (i) a dedicated ionization chamber for electron beam dosimetry (Advance Markus type) to monitor the total applied dose for each experiment; (ii) three TLDs (Panasonic type) to monitor the dose through the cell containers; and (iii) a Gafchromic film (GF-EBT3 type) to provide correlation with the dose distribution on the microscopic slide (Fig.  7 ). All dosimetry systems were calibrated before the experiment. The TLD detectors were prepared and read by an accredited third-party laboratory.

Schematic representation of the pulsed X-ray irradiation setup.

An Advance Markus ionization chamber type TN34045, which is an air-vented plane-parallel ionization chamber, was used to measure the total dose absorbed by water in electron beams. Compared with other chambers, the Advance Markus chamber demonstrated the best performance for the FLASH doses 7 . The chamber was connected to a UNIDOS dosimeter from PTW. Measurements were performed with a continuous charge collection low-dose regime.

The TLD Panasonic thermoluminescent dosimeters contained 4 thermoluminescent elements (two of Li 2 B 4 O 7 : Cu (with Z efectiv close to human tissue) and two of CaSO 4 : Tm (high sensitivity)) to determine the average radiation energy and the dose recorded by the dosimeter, using all the information provided by the 4 elements. A digital thermo-luminescent dosimeter (TL Badge) (model Panasonic UD-716) was used as the reader. The sample stack was perpendicular to the electron beam axis. The relative depth doses were measured. No beam collimator was used.

The Gafchromic film was a self-developed EBT-3 designed for the measurement of absorbed doses of ionizing radiation. These films are widely used in medical radiotherapy departments for quality assurance and dose measurements. The film darkening was measured using an integrated densitometer in a dedicated EPSON Expression 11000XL professional scanner with a resolution of 4800 dpi in transmission mode 61 .

Pulsed X-ray setup

The setup used for delivering pulsed X-rays consisted of an X-ray chopper made of steel with a brass infusion, which had 12 slits (each with a height of 8 mm and a width of 1.5 mm) placed at 15-degree intervals from each other. It also included a sample holder that positioned the sample at a distance of 10 cm from the head of the X-ray source. A pair of lead collimators, each 2 mm thick, was placed in front of the X-ray head to reduce the size of the X-ray beam and X-ray scattering. The chopper was rotated by a high-speed brushless DC motor connected to an electronic speed controller (ESC) unit. The rotational speed was controlled with the help of an electronic board that provided a pulse width modulated (PWM) signal. During the irradiation experiments, the servo position was set with a pulse width of 1469 µs, which corresponded to an X-ray pulse frequency of 1.4 kHz. The X-ray source was set to a voltage of 200 kV and a current of 100 µA, and a copper foil with a thickness of 0.2 mm was placed in front for radiation filtration. For these X-ray source operating parameters, under continuous exposure, a dose rate of 26 mGy/s at a distance of 10 cm was measured by an ionization chamber connected to a standard UNIDOS dosimeter. In the case of pulsed X-ray irradiation conditions, the doses delivered to the samples were estimated using Gafchromic EBT3 films that were placed at the back of the cell culture Slide Flask (Fig.  7 ). The samples were irradiated with pulsed X-rays for 15 min and 31 s, and according to Gafchromic EBT3 measurements, the irradiated flask region (symbolled with a T sign in Fig.  7 ) received a total dose of 2.4 Gy. The EBT3 films were previously irradiated with precisely delivered doses, and calibration curves were determined from the radiochromic film darkening and were proportional to the absorbed dose. The setup for pulsed X-ray irradiation was kept identical between irradiation campaigns.

Quantification of the biological effects

After irradiation (at the PW laser or X-ray facilities) at room temperature (RT), the cells were allowed to recover in the incubator for 30 min and then washed once with phosphate-buffered saline (PBS) before being fixed together with the controls using 4% p-formaldehyde (PFA) for 30 min at RT. The cells were washed twice with PBS and stored at 4 °C until further processing.

To evaluate the double-strand DNA breaks in response to radiation exposure, we examined the appearance of the nuclear p-γ-H2AX foci using specific mouse monoclonal antibodies for the Ser140 phosphorylated form of γ-H2AX (#MA1-2022, Invitrogen, Thermo). The fixed cells were first permeabilized using 0.02% Triton-X-100 in PBS for 3 min, followed by 3 washes in PBS. Next, 0.5% BSA-PBS was added for a 1 h incubation to block nonspecific binding sites. The antibody was used at a 1:200 dilution in the blocking buffer for 30 min at RT, followed by a series of PBS washes and incubation with AlexaFluor 594-conjugated donkey anti-mouse secondary antibodies (#A21203, Invitrogen, Thermo). Finally, the nuclei were labelled with Hoechst 33342 (#H21491, Molecular Probes, Thermo) for 1 min, and after thorough washing, the samples were mounted using FluorSave Reagent (#345789, Millipore) and left to dry overnight. The slides were scanned using the TissueFAXSiPlus automated imaging system (TissueGnostics, Vienna, Austria), and the manual focus option was selected to generate the optimal fluorescence images given the planarity imperfections of the SlideFlask polystyrene surface. Stitched overview images were generated for each slide during previewing at 5 × magnification. The regions of interest (ROIs) were created using the rectangle tool for each area to be investigated, considering irradiation areas of interest; similar ROIs were delineated on controls. Next, ROIs were obtained using a 63 × oil objective, excluding the environmental controls; the environmental controls were examined using 20 × air due to the objective working distance limitations for focusing into the 24-well plate bottom.

Image cytometry analysis

To determine the percentage of p-γ-H2AX + cells, the mean fluorescence intensity (MFI) of the p-γ-H2AX signal, and the number of p-γ-H2AX foci per nucleus, the samples were scanned with the TissueFAXS Slides 3.5.5.0129 module using a 63 × oil objective. The signals recorded through the DAPI (λ ex 360 nm/λ em 462 nm) and TxRed (λ ex 568 nm/ λ em 603 nm) filters were further analysed upon nuclear segmentation using TissueQuest software version n. 4.0.1.0140 (TissueGnostics, Vienna, Austria, URL: https://tissuegnostics.com/products/single-cell-analysis/tissuequest ,). Using the gating strategy exemplified in Fig.  8 for the CisPt positive control 62 , DAPI-area, and DAPI-mean intensity parameters were used for single cell recognition, followed by DAPI-compactness vs. DAPI-mean intensity to select compact nuclei and exclude irregularly shaped artefacts. Next, the gated compact nuclei were visualized in a DAPI eccentricity vs. DAPI-mean intensity scattergram to identify elongated nuclei (high eccentricity) of the NHEM cells and non-elongated nuclei (low eccentricity) of the A375 cells. The Texa-Mean Intensity and Texa_DOTS_ON_DAPI parameters were used to quantify the p-γ-H2AX MFI (Figure SI 4 fig) and the number of foci (Fig.  4 ), respectively.

Gating strategy used for image cytometry analysis in TissueQuest 4.0.1.0140 for cells exposed to different irradiation conditions. Scattergrams of cisplatin (CisPt)-treated positive control co-cultures are given here as examples. CisPt is a known inducer of DNA damage and is used to treat various types of cancers.

The thresholds for every parameter were optimized using backward gating to examine the images of the segmented cells within each population. Additionally, the setup was validated using negative control samples (untreated, non-irradiated). Statistical reports and dot statistics were generated to extract the quantification values for each sample under investigation. A total number of 7370–15,739 cells were contained in each condition, depending on each region of interest (ROI) cellular content. Upon removal of artefacts by drawing exclusion regions and filtering of data through sequential gating (Fig.  8 , first 3 scattergrams), the number of nuclei left for p-γ-H2AX quantification were between 4473 and 10,629 (see Table SI 1 ).

Statistical data analysis

Experimental data are reported of two serial replicates (PW1 and PW2) from one irradiation session out of two performed. The image cytometry data were imported into GraphPad Prism 9.5.1 (528) to generate bar graphs of the obtained values and to analyse the statistical differences between the PW or X-ray irradiation conditions, and the control non-irradiated sample. To evaluate potential differences in double-strand DNA breaks induction, statistical comparisons were performed at 30 min post-irradiation by Mann–Whitney test. A p value of < 0.05 was considered to indicate statistical significance. All adjusted p values are represented on the graphs as star symbols and explained in figure legends.

Data availability

All data are available within the Article and Supplementary Files or available from the corresponding authors upon reasonable request.

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Acknowledgements

This research was supported by IFA (Institute of Atomic Physics) through the ELI-RO_2020_11 Project, No. 01/2020; UEFISCDI through the PCE Project, No. 8/2021; and the Romanian Ministry of Education and Research under the Romanian National Nucleus Program LAPLAS VII under Contract No. 30N/2023. This work received funding from the European Union’s Horizon 2020 Research and Innovation Program under Grant Agreement No. 871124 Laserlab-Europe. S.O. and L.E.S. acknowledge the partial support of Project No. 1/2023 of the Research Program of the Institute of Biochemistry of the Romanian Academy. We acknowledge the support of National Interest Infrastructure facility IOSIN – CETAL at INFLPR. The experiments presented here are the first to confirm the DNA damage-producing capacity of the laser-driven high-energy-dose electrons produced by the CETAL-PW laser infrastructure.

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These authors contributed equally: Stefana Orobeti, Livia Elena Sima.

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National Institute for Laser, Plasma and Radiation Physics (INFLPR), 409 Atomistilor Street, RO-077125, Magurele, Romania

Stefana Orobeti, Ioana Porosnicu, Constantin Diplasu, Georgiana Giubega, Gabriel Cojocaru, Razvan Ungureanu, Cosmin Dobrea, Mihai Serbanescu, Alexandru Mihalcea, Elena Stancu, Cristina Elena Staicu, Florin Jipa, Alexandra Bran, Emanuel Axente, Simion Sandel, Marian Zamfirescu, Ion Tiseanu & Felix Sima

Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, 060031, Bucharest, Romania

Stefana Orobeti & Livia Elena Sima

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Contributions

F.S. and I.T. conceived and coordinated the project. L.E.S., I.T., E.A., M.Z., S.S., and F.S. co-supervised the study. S.O. contributed to the optimization of the setup for cell irradiation and performed IF and scanning of the resulting specimens. L.E.S. processed the images and analysed the experimental data and results. C.D., G.G., G.C., R.U., C.D., M.S., A.M., C.E.S., F.J., and A.B. optimized the experimental protocols and prepared the LPA experimental setup for cell exposure. IP and IT optimized and prepared the pulsed X-ray setup and completed the theoretical analysis and numerical simulations. E.S. performed the dosimetry analyses. F.S. and L.E.S. drafted the manuscript. All the authors contributed to the data interpretation and finalized/revised the manuscript.

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Correspondence to Felix Sima .

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Orobeti, S., Sima, L.E., Porosnicu, I. et al. First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies. Sci Rep 14 , 14866 (2024). https://doi.org/10.1038/s41598-024-65137-7

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Received : 24 January 2024

Accepted : 17 June 2024

Published : 27 June 2024

DOI : https://doi.org/10.1038/s41598-024-65137-7

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